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<div id='write' class = ''><p><img src="./images/MRC_Logo.PNG" referrerpolicy="no-referrer" alt="MRC LOGO"></p><h1><a name="mrc-microbiome-analysis-toolbox-a-singularity-container-version" class="md-header-anchor"></a><span>MRC microbiome analysis toolbox-A singularity container version </span></h1><p><span>Prepared by MRC bioinformatics team </span><strong><span>Dr Xiao-Tao Jiang</span></strong><span>, </span><strong><span>Dr Emily McGovern</span></strong><span> and </span><strong><span>Dr Fan Zhang</span></strong></p><p><span>Date: 2020-09-15</span></p><p><span>Due to complex dependency and version conflicts, bioinformaticians are strugglying about installation of different tools and solving their dependence relationship and debuging while version conflicts happens. To solve this problem and establish a comprehensive, mobile and easy to use framwork for microbiome analysis to beginners, we build this MRC microbiome singularity image. This document describe the interface for different aspects of analysis using a demo example and the mrcmicrobiome.sif singularity image. </span></p><!--ts--><ul><li><p><a href='#mrc-microbiome-analysis-toolbox-a-singularity-container-version'><span>MRC microbiome analysis toolbox-A singularity container version</span></a></p></li><li><p><a href='#16s-amplicon-data-analysis'><span>16S amplicon data analysis</span></a></p><ul><li><a href='#prerequisites'><span>Prerequisites</span></a></li></ul></li><li><p><a href='#structure'><span>Structure</span></a></p></li><li><p><a href='#schedule'><span>Schedule</span></a></p></li><li><p><a href='#1-linux-and-hpc-setup'><span>1. Linux and HPC setup</span></a></p><ul><li><a href='#overviewoverviewoverviewoverviewoverviewoverviewoverviewoverviewoverviewoverviewoverview'><span>Overview</span></a></li><li><a href='#katana--unsw-research'><span>Katana | UNSW Research</span></a></li><li><a href='#log-on-to-katana'><span>Log on to Katana</span></a></li><li><a href='#working-on-katana'><span>Working on Katana</span></a></li><li><a href='#navigating-katana'><span>Navigating Katana</span></a></li><li><a href='#data-upload'><span>Data upload </span></a></li><li><a href='#futher-navigation-of-katana'><span>Futher Navigation of Katana</span></a></li><li><a href='#further-training'><span>Futher Training</span></a></li><li><a href='#reference-linux-commands'><span>Reference Linux commands</span></a></li><li><a href='#text-editing-from-the-command-line'><span>Text editing from the command line</span></a></li></ul></li><li><p><a href='#2-quality-control'><span>2. Quality control</span></a></p><ul><li><a href='#understand-the-demo-data-set'><span>Understand the demo data set</span></a>
<span> * </span><a href='#overview-1'><span>Overview</span></a></li><li><a href='#singularity-on-katana'><span>Singularity on katana</span></a></li><li><a href='#check-the-demo-and-database-db-directory'><span>Check the demo and database db directory</span></a></li><li><a href='#qiime2-manifest-file-preparation-example-below'><span>QIIME2 manifest file preparation, example below</span></a></li><li><a href='#prepare-manifest-and-run-dada2'><span>Prepare manifest and run dada2</span></a>
<span> * </span><a href='#overview-2'><span>Overview</span></a></li><li><a href='#overall-assessment-of-quality-of-sequences-with-fastp'><span>Overall assessment of quality of sequences with fastp</span></a></li><li><a href='#import-fasta-sequences-into-demuxqza'><span>Import fasta sequences into demux.qza</span></a></li><li><a href='#running-dada2-within-qiime2-to-perform-quality-control-this-takes-about-1-minute'><span>Running dada2 within qiime2 to perform quality control, this takes about 1 minute</span></a></li><li><a href='#host-contamination-removal-if-necessary-when-processing-mucosal-samples'><span>Host contamination removal if necessary when processing mucosal samples</span></a>
<span> * </span><a href='#overview-3'><span>Overview</span></a></li></ul></li><li><p><a href='#3-taxonomy-annotation-and-diversity-analysis'><span>3. Taxonomy annotation and diversity analysis</span></a></p><ul><li><p><a href='#overview-4'><span>Overview</span></a></p></li><li><p><a href='#taxonomy-assignment-of-feature-sequences'><span>Taxonomy assignment of feature sequences</span></a>
<span> * </span><a href='#overview-5'><span>Overview</span></a></p></li><li><p><a href='#phylogenetic-tree-construction'><span>Phylogenetic tree construction</span></a>
<span> * </span><a href='#overview-6'><span>Overview</span></a></p></li><li><p><a href='#run-the-core-diversity-analysis'><span>Run the core diversity analysis</span></a>
<span> * </span><a href='#overview-7'><span>Overview</span></a></p></li><li><p><a href='#alpha-diversity-rarefaction-curve'><span>Alpha diversity rarefaction curve</span></a>
<span> * </span><a href='#overview-8'><span>Overview</span></a></p></li><li><p><a href='#permanova-statistical-testing-of-alpha-and-beta-diversity-with-meta-data'><span>PERMANOVA statistical testing of alpha and beta diversity with meta data</span></a>
<span> * </span><a href='#overview-9'><span>Overview</span></a></p><ul><li><a href='#permanova-statistical-testing-of-alpha-diversity-for-groups'><span>PERMANOVA statistical testing of alpha diversity for groups</span></a></li><li><a href='#permanova-statistical-testing-of-beta-diversity-for-groups'><span>PERMANOVA statistical testing of beta diversity for groups</span></a></li></ul></li></ul></li><li><p><a href='#4-using-lefse-to-identify-differential-abundant-taxa'><span>4. Using LEfSe to identify differential abundant taxa</span></a></p><ul><li><a href='#overview-10'><span>Overview</span></a></li><li><a href='#cladoplot-presenting-of-signatures'><span>cladoplot presenting of signatures</span></a></li><li><a href='#lefse-signatures-barchart'><span>LEfSe signatures barchart</span></a></li></ul></li></ul><!-- Added by: z3524677, at: Wed Sep 23 19:33:10 AEST 2020 --><!--te--><h1><a name="16s-amplicon-data-analysis" class="md-header-anchor"></a><span>16S amplicon data analysis </span></h1><p><span>In this tutorial, we use singularity version of the pipeline </span><strong><em><span>mrcmircobiome.sif</span></em></strong><span> to run a high fat diet mouse model stool microbiome analysis. The demo dataset includes stools samples from mice on divergent diets: high fat diet v normal chow. Using this demo dataset, users will learn:</span></p><ol start='' ><li><span>How to prepare data for analysis</span></li><li><span>Quality control of amplicon sequences</span></li><li><span>Taxonomic annotation of 16S rRNA sequences</span></li><li><span>Alpha and beta diversity analysis </span></li><li><span>Differential abundant analysis using Linear discriminant analysis Effect Size </span><a href='https://github.com/biobakery/biobakery/wiki/lefse'><span>LEfSe</span></a></li></ol><blockquote><h2><a name="prerequisites" class="md-header-anchor"></a><span>Prerequisites</span></h2><p><span>You’ll need a laptop with a web browser and a terminal. See also the </span><a href='https://'><span>Setup</span></a><span> page.</span></p><p><span>You'll need a katana account for the training/have the the singularity file </span><strong><em><a href=''><span>mrcmicrobiome.sif</span></a></em></strong><span> (This file is already on katana)</span></p><p><span>You might want to brush up on the fundamentals of the </span><a href='https://'><span>Linux Shell for HPC</span></a><span>.</span></p></blockquote><h1><a name="structure" class="md-header-anchor"></a><span>Structure</span></h1><p><span>This tutorial has been designed in a modular way. Modules are categorised as follows:</span></p><ul><li><span>Basic linux and setup prerequest environment</span></li><li><span>16S microbiome analysis: taxonomy, alpha, beta diversity analysis and basic statistical analysis </span></li><li><span>Microbial signatures identification with LEfSe</span></li></ul><p><span>Once you’ve gone through this tutorial, you will be able to analyze 16S amplicon sequences microbiome data</span>
<span>You can refer to </span><a href='https://docs.qiime2.org/2020.8/'><span>QIIME2</span></a><span>, </span><a href='http://benjjneb.github.io/dada2/tutorial.html'><span>DADA2</span></a><span>, </span><a href='https://mothur.org/'><span>Mothur</span></a><span>, </span><a href='https://www.drive5.com/uparse/'><span>UPARSE</span></a><span> and </span><a href='https://joey711.github.io/phyloseq/'><span>Phyloseq</span></a><span> for more information</span></p><h1><a name="schedule" class="md-header-anchor"></a><span>Schedule</span></h1><figure><table><thead><tr><th> </th><th><a href='https://'><span>Setup</span></a></th><th> </th></tr></thead><tbody><tr><td><span>10:00 - 10:30 am</span></td><td><span>1. </span><a href='http://'><span>Linux and HPC setup and data transfer</span></a></td><td> </td></tr><tr><td><span>10:40 - 12:00 am</span></td><td><span>2. </span><a href='https://'><span>Introduce of demo data set and quality control</span></a></td><td> </td></tr><tr><td><span>13:30 - 14:30 Pm</span></td><td><span>3. </span><a href='https://'><span>Taxonomy, phylogenetic tree, alpha and beta diversity analysis</span></a></td><td> </td></tr><tr><td><span>14:50 - 15:30 Pm</span></td><td><span>4. </span><a href='https:'><span>LEfSe to identify microbial signatues</span></a></td><td> </td></tr></tbody></table></figure><p><span>The actual schedule may vary slightly depending on the topics and exercises chosen by the instructor.</span></p><p> </p><h1><a name="1-linux-and-hpc-setup" class="md-header-anchor"></a><span>1. Linux and HPC setup </span></h1><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 30 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Learn basic linux commands and HPC </span></li><li><span>Learn how to transfer data from HPC to local computers</span></li></ul><h2><a name="katana--unsw-research" class="md-header-anchor"></a><span>Katana | UNSW Research </span></h2><p><span>Katana is a shared computational cluster located on campus at UNSW that has been designed to provide easy access to computational resources for groups working with non-sensitive data. Katana will be used for your next generation sequencing analysis.</span></p><h2><a name="log-on-to-katana" class="md-header-anchor"></a><span>Log on to Katana</span></h2><h3><a name="log-on-from-iterm-mac" class="md-header-anchor"></a><span>Log on from Iterm (Mac)</span></h3><p><span>Download a terminal in order to log on to a remote terminal e.g. </span><a href='https://www.iterm2.com/'><span>Iterm</span></a><span> (Mac) or </span><a href='https://mobaxterm.mobatek.net/'><span>mobaxterm</span></a><span> (Windows)</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash,eval=F, echo=T}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash,eval=f, echo=t}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">ssh zID@katana.restech.unsw.edu.au</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><img src="./images/katana_logon.png" style="zoom:90%;" /></p><h3><a name="log-on-from-mobaxterm-windows" class="md-header-anchor"></a><span>Log on from Mobaxterm (Windows)</span></h3><p><img src="./images/mobaxterm_windows.png" style="zoom:90%;" /></p><h2><a name="working-on-katana" class="md-header-anchor"></a><span>Working on Katana</span></h2><p><span>Always create an </span><a href='https://research.unsw.edu.au/katana-compute-jobs'><span>interactive job</span></a><span> to work on Katana - Do not run jobs on head node</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash,eval=F, echo=T}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash,eval=f, echo=t}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">qsub -I -q R717942 -l nodes=1:ppn=1,mem=10gb,walltime=6:00:00</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><img src="./images/interactive_session.png" style="zoom:90%;" /></p><h2><a name="navigating-katana" class="md-header-anchor"></a><span>Navigating Katana</span></h2><p><span>Check your filepath</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">pwd</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Move into your personal scratch directory. Always create an </span><a href='https://research.unsw.edu.au/katana-compute-jobs'><span>interactive job</span></a><span> to work on Katana - Do not run jobs on head node</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> /srv/scratch/zID</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>List what is in your filepath</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">ls</span></span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Make a new directory called </span><code>bio_tutorial</code></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">mkdir</span> bio_tutorial</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Move into the </span><code>bio_tutorial</code><span> directory </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> bio_tutorial</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Move back a directory </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> ..</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><h2><a name="data-upload" class="md-header-anchor"></a><span>Data upload </span></h2><p><span>On your desktop and open notepad and create a file with the following text and save to your desktop as </span><code>test_file.txt</code></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-attribute">-8ACCGGB</span>@<span class="cm-operator">=</span><span class="cm-number">8</span><span class="cm-operator">=</span>6BCFFCDEFGFGGGGGGGGGGGGGGFFGF@C@CFGGGFCCBFGGGFGCFGGGFGDCFGGFGGGGGFFCGFFGDFCGGCGGCC<EBFFGFBBE<EFFGEEFGG?<F9?E7<span class="cm-operator">++</span><span class="cm-number">8</span><span class="cm-operator">+</span>,,:<?<,<span class="cm-operator">=++</span>@:FEGGD9F@7F7><span class="cm-operator">=</span>F;8FFG@7FFCFCFFCCBCF8>DFCFEBFGGFGGDC:F:*<F7<F,4<B9C*CCCACEFFEGC:EE:CE*<span class="cm-def">*A89CFCFCC</span><span class="cm-operator">=</span>C5**<<span class="cm-operator">+</span><span class="cm-number">55</span>;C<span class="cm-def">*CCE</span><span class="cm-operator">=</span>EC>C58?F7:@7<:<><span class="cm-operator">+</span><span class="cm-number">37</span>:C7C99@E77C:<C*;<span class="cm-operator">++</span><span class="cm-number">8</span>:735B))8@36>>BC?)</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">@M01153:567:000000000-CNVPV:1:1101:23398:1773 <span class="cm-number">1</span>:N:0:GCTCATGA<span class="cm-operator">+</span>GCGTAAGA</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">CCTACGGGGGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGACGTTAGTAACTGAACGTCCCCTGACGGTATCTAACCAGACAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCCAGCGTTGTCCGGTTTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGTT</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><span>Next let's upload this file to your scratch drive on Katana using </span><strong><span>Katana data Mover</span></strong></p><h3><a name="open-filezilla" class="md-header-anchor"></a><span>Open FileZilla</span></h3><ol start='' ><li><span>Enter the following details in the top tool bar</span></li></ol><ul><li><span>Hostname = </span><code>ssh zID@kdm.restech.unsw.edu.au</code></li><li><span>Username = zID</span></li><li><span>Password = zID password</span></li><li><span>Port = 22</span></li></ul><p><img src="./images/FileZilla_restech.PNG" style="zoom:90%;" /></p><h3><a name="enter-remote-site-filepath" class="md-header-anchor"></a><span>Enter remote site filepath</span></h3><ol start='2' ><li><span>Once your logged on add </span><code>/srv/scratch/zID</code><span> to the bar saying </span><code>remote site</code></li></ol><h3><a name="uploading-file" class="md-header-anchor"></a><span>Uploading file</span></h3><p><span>Copy </span><code>test_file.txt</code><span> into your </span><code>/srv/scratch/zID</code></p><h2><a name="futher-navigation-of-katana" class="md-header-anchor"></a><span>Futher Navigation of Katana</span></h2><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cp</span> test_file.txt bio_tutorial/</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Move into </span><code>bio_tutorial</code><span> directory </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> bio_tutorial/</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Now lets list whats in the </span><code>bio_tutorial</code><span> directory </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">ls</span> </span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Duplicate the file </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cp</span> test_file.txt test_file2.txt</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>List all files with the suffix </span><code>.txt</code></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">ls</span> *.txt</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Remove test_file2.txt</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">rm</span> test_file2.txt</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Look inside test_file.txt</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">less test_file.txt</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Move back into scratch directory</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> ..</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>Delete </span><code>bio_tutorial</code></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="{bash, eval=FALSE}"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="{bash, eval=false}"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">rm</span> <span class="cm-attribute">-r</span> bio_tutorial/</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><h2><a name="further-training" class="md-header-anchor"></a><span>Further Training </span></h2><p><span>UNSW research technology offer a range of training courses, that as of 2020 have been moved online. These are a great introduction to general programming and will help you improve the efficiency of your work. Explore them </span><a href='https://research.unsw.edu.au/research-technology-training'><span>here</span></a><span> </span></p><p><span>Introduce yourself to basic linux command using an </span><a href='http://www.ee.surrey.ac.uk/Teaching/Unix/'><span>online course</span></a></p><h2><a name="reference-linux-commands" class="md-header-anchor"></a><span>Reference Linux commands</span></h2><p><span>Use this table as a reference during the workshop</span></p><figure><table><thead><tr><th><span>Command</span></th><th><span>Meaning</span></th><th><span>Example</span></th></tr></thead><tbody><tr><td><code>ls</code></td><td><span>list files in current directory, with -l, it also displays file permissions, sizes and last updated date/time</span></td><td><code>ls -l</code><span>, </span><code>ls</code></td></tr><tr><td><code>pwd</code></td><td><span>displays your current location in the file system</span></td><td><code>pwd</code></td></tr><tr><td><code>env</code></td><td><span>displays your user environment settings (e.g. search path, history size and home directory)</span></td><td><code>env</code></td></tr><tr><td><code>cd</code></td><td><span>change directory</span></td><td><code>cd /file/path</code></td></tr><tr><td><code>mv</code></td><td><span>move file, change file name - </span><strong><span>Careful with this, no undo!</span></strong></td><td><code>mv /path/file/ /newpath/file</code><span> , </span><code>mv oldname newname</code></td></tr><tr><td><code>cp</code></td><td><span>copy files, -R copy directory</span></td><td><code>cp /path/file/ /newpath/file</code><span>, </span><code>cp -R ./directory ./newdirectorylocation</code></td></tr><tr><td><code>head</code></td><td><span>print top lines in file</span></td><td><code>head filename</code><span> , </span><code>head -10 filename</code></td></tr><tr><td><code>tail</code></td><td><span>print last lines in file</span></td><td><code>tail filename</code><span> , </span><code>tail -10 filename</code></td></tr><tr><td><code>less</code></td><td><span>view lines in file, use spacebar to go down file</span></td><td><code>less filename</code></td></tr><tr><td><code>grep</code></td><td><span>search file for pattern or string</span></td><td><code>cat filename |grep "aaatttcc"</code></td></tr><tr><td><code>*</code></td><td><span>wildcard, use with other commands</span></td><td><code>ls *.fastq</code><span> : list all files with suffix ".fastq"</span></td></tr><tr><td><code>wc</code></td><td><span>word count, with -l = linecount</span></td><td><code>wc filename</code><span>, </span><code>wc -l filename</code></td></tr><tr><td><code>rm</code></td><td><span>remove filename , with -R directory </span><strong><span>Again careful</span></strong></td><td><code>rm filenmae</code></td></tr><tr><td><code>exit</code></td><td><span>exit remote server</span></td><td><code>exit</code></td></tr><tr><td><code>.</code></td><td><span>This represents the current directory</span></td><td><code>pwd .</code><span>, </span><code>cd ./path/to/subdirectory/from/currentdir</code></td></tr><tr><td><code>..</code></td><td><span>This represents the parent directory</span></td><td><code>pwd ..</code><span>, </span><code>cd ../path/to/subdirectory/from/parentdir</code></td></tr></tbody></table></figure><p><span>Table:Useful everyday commands</span></p><h2><a name="text-editing-from-the-command-line" class="md-header-anchor"></a><span>Text editing from the command line</span></h2><p><span>When learning bioinformatics, you will most likely need to create or edit text files, shell scripts or Python scripts from the command line. Using a Unix-based text editor good practice for getting used to the environment if you are new to the command line. </span></p><p><span>There are multiple text editors available. Which one you use is based on user preference, beginners usually go for nano or vim.</span></p><p><strong><span>Text Editors</span></strong></p><ul><li><span>nano</span></li><li><span>vim</span></li><li><span>emacs</span></li></ul><p><strong><span>User Guides</span></strong></p><ul><li><span>Simple </span><a href='https://rachaellappan.github.io/learn-vim/'><span> guide</span></a><span> from a beginners perspective</span></li><li><span>More comprehensive </span><a href='https://bioinformatics.uconn.edu/vim-guide/'><span>guide</span></a><span> to vim</span></li></ul><p><strong><span>Useful Tip</span></strong><span> </span></p><p><span>If using </span><code>vim</code><span> and you are stuck in a wrong mode etc, you can always escape using by pressing </span><code>ESC</code><span> followed by </span><code>:q!</code><span> and this will allow you exit without saving any changes.</span></p><h1><a name="2-quality-control" class="md-header-anchor"></a><span>2. Quality control</span></h1><h2><a name="understand-the-demo-data-set" class="md-header-anchor"></a><span>Understand the demo data set</span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Download the demo data set</span></li><li><span>Understand the meta data file and learn how to prepare it</span></li></ul><p><span>Using the FTP software to upload the demo data set and the relevant meta data file to your HPC account</span></p><h2><a name="singularity-on-katana" class="md-header-anchor"></a><span>Singularity on katana</span></h2><p><span>login to compute nodes to run jobs. </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">qsub <span class="cm-attribute">-I</span> <span class="cm-attribute">-q</span> R717942 <span class="cm-attribute">-l</span> <span class="cm-def">nodes</span><span class="cm-operator">=</span><span class="cm-number">1</span><span class="cm-def">:ppn</span><span class="cm-operator">=</span><span class="cm-number">1</span><span class="cm-def">,mem</span><span class="cm-operator">=</span>10gb<span class="cm-def">,walltime</span><span class="cm-operator">=</span><span class="cm-number">6</span>:00:00</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>mrcmicrobiome singularity sif have conda, qiime2-2020.8 and lefse installed, all the relevant dependence are resolved. Copy the demo data set to your own work directory </span></p><p><span>Check the singularity image file /data/bio/workshop/mrcmicrobiome.sif </span></p><p><span>Using the following command to create the wrok directory and copy the demo data set to your directory</span>
<strong><span>Remember to replace the yourzID with your zID</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><span><span></span>x</span></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#create the hfdiet working directory </span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">mkdir</span> /srv/scratch/yourzID/hfdiet</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#enter into the directory</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> /srv/scratch/yourzID/hfdiet</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#copy the demo data set into your work directory</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cp</span> <span class="cm-attribute">-r</span> /data/bio/workshop/demo/ /srv/scratch/yourzID/hfdiet</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 176px;"></div><div class="CodeMirror-gutters" style="display: none; height: 176px;"></div></div></div></pre><h2><a name="check-the-demo-and-database-db-directory" class="md-header-anchor"></a><span>Check the demo and database db directory</span></h2><p><span>There are in total 16 samples in the demo, this demo compare mice stool microbiome with normal and high fat diet chow check the </span><strong><span>meta_data.txt</span></strong><span> file, you can find the samples information </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 hfdiet]<span class="cm-def">$ tree</span> demo/</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">demo/</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- all_r1.fq</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- all_r2.fq</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- meta_data.txt</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-quote">`-- rawfq</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5821_S50_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5821_S50_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5822_S34_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5822_S34_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5824_S96_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5824_S96_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5825_S7_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5825_S7_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5832_S8_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5832_S8_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5833_S78_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5833_S78_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5859_S27_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5859_S27_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5860_S122_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5860_S122_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5862_S51_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5862_S51_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5863_S30_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 5863_S30_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6319_S128_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6319_S128_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6320_S67_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6320_S67_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6321_S20_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6321_S20_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6322_S131_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6322_S131_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6328_S93_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6328_S93_L001_R2.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> |-- 6329_S37_L001_R1.fastq.gz</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> <span class="cm-quote">`-- 6329_S37_L001_R2.fastq.gz</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> </span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 902px;"></div><div class="CodeMirror-gutters" style="display: none; height: 902px;"></div></div></div></pre><p><span>Check meta data file </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#enter into your own directory </span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> demo/</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#show the contents of the meta data file </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ cat</span> meta_data.txt</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-comment">#SampleID<span class="cm-tab" role="presentation" cm-text=" "> </span>LANID<span class="cm-tab" role="presentation" cm-text=" "> </span>Diet</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6319_S128_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>fvb-a<span class="cm-tab" role="presentation" cm-text=" "> </span>Normal</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6320_S67_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>fvb-a<span class="cm-tab" role="presentation" cm-text=" "> </span>Normal</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6321_S20_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>fvb-b<span class="cm-tab" role="presentation" cm-text=" "> </span>Normal</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6322_S131_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>fvb-b<span class="cm-tab" role="presentation" cm-text=" "> </span>Normal</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6328_S93_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>fvb-c<span class="cm-tab" role="presentation" cm-text=" "> </span>Normal</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6329_S37_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>fvb-c<span class="cm-tab" role="presentation" cm-text=" "> </span>Normal</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5821_S50_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-1</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5822_S34_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-1</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5824_S96_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-2</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5825_S7_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-2</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5832_S8_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-3</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5833_S78_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-3</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5859_S27_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-4</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5860_S122_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-4</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5862_S51_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-5</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5863_S30_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">181</span><span class="cm-attribute">-5</span><span class="cm-tab" role="presentation" cm-text=" "> </span>HFD</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 484px;"></div><div class="CodeMirror-gutters" style="display: none; height: 484px;"></div></div></div></pre><p><span>Check the database folder </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">tree /data/bio/workshop/db/</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">/data/bio/workshop/db</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- gg-v3v4-classifier.qza</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- gg-v4-classifier.qza</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- mouse.genome.bowtie2.1.bt2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- mouse.genome.bowtie2.2.bt2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- mouse.genome.bowtie2.3.bt2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- mouse.genome.bowtie2.4.bt2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- mouse.genome.bowtie2.rev.1.bt2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-quote">`-- mouse.genome.bowtie2.rev.2.bt2</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 264px;"></div><div class="CodeMirror-gutters" style="display: none; height: 264px;"></div></div></div></pre><h2><a name="qiime2-manifest-file-preparation-example-below" class="md-header-anchor"></a><span>QIIME2 manifest file preparation, example below </span></h2><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">sample-id,absolute-filepath,direction</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5821_S50_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5821_S50_L001_R1.fastq.gz,forward</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5821_S50_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5821_S50_L001_R2.fastq.gz,reverse</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5822_S34_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5822_S34_L001_R1.fastq.gz,forward</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5822_S34_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5822_S34_L001_R2.fastq.gz,reverse</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5824_S96_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5824_S96_L001_R1.fastq.gz,forward</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5824_S96_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5824_S96_L001_R2.fastq.gz,reverse</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5825_S7_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5825_S7_L001_R1.fastq.gz,forward</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5825_S7_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5825_S7_L001_R2.fastq.gz,reverse</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5832_S8_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5832_S8_L001_R1.fastq.gz,forward</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5832_S8_L001,/srv/scratch/z3524677/hfdiet/demo/rawfq//5832_S8_L001_R2.fastq.gz,reverse</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 242px;"></div><div class="CodeMirror-gutters" style="display: none; height: 242px;"></div></div></div></pre><h2><a name="prepare-manifest-and-run-dada2" class="md-header-anchor"></a><span>Prepare manifest and run dada2</span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Generate quality control shell scripts with </span><strong><em><span>mima_prepare_manifest_and_qc.pl</span></em></strong></li><li><span>Understand how to perform quality control with dada2</span></li></ul><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec <span class="cm-attribute">--cleanenv</span> /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'perl /home/applications/mima/mima_prepare_manifest_and_qc.pl'</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">perl /home/applications/mima/mima_prepare_manifest_and_qc.pl <Absolute path to store fastq files> <output.manifest> <total_1.fq> <total_2.fq> <outputdir> <<span class="cm-comment"># of threads> <Singularity_sif></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><inputdir_abs_path> The absolute directory to store the pair-end fastq files</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><output.manifest> The manifest file that qiime needed when running dada2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><Merged_1.fq> The overall fastq file pair <span class="cm-number">1</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><Merged_2.fq> The overall fastq file pair <span class="cm-number">2</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><Output_dir> Output directory store all the QC files and dada2 output</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><<span class="cm-comment">#ofthreads><span class="cm-tab" role="presentation" cm-text=" "> </span> Deafault 1</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><Singularity sif img> the singualrity image to use</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 286px;"></div><div class="CodeMirror-gutters" style="display: none; height: 286px;"></div></div></div></pre><p><span>Run the one comman pipeline to generate all the necessary scripts. This one command will generate all the necessary scripts for the QC analysis. You can submit this as a job or run each command individually in an interactive job. As QC can take time, it's advisable to submit all the commands as a job </span><code>qsub qc.pbs</code><span>. For learning purposes today we will go through each command and it's output in an interactive model as the demo small</span></p><p><strong><span>Remember to change the zID to your own zID</span></strong><span> </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#run the command to generate commands for quality control </span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#Absolute path should be used input and output directory </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec <span class="cm-attribute">--cleanenv</span> /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'perl /home/applications/mima/mima_prepare_manifest_and_qc.pl /srv/scratch/z3524677/hfdiet/demo/rawfq/ /srv/scratch/z3524677/hfdiet/demo/hf_d.manifest all_r1.fq all_r2.fq /srv/scratch/z3524677/hfdiet/demo/hf_qc 1 /data/bio/workshop/mrcmicrobiome.sif'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ tree</span> hf_qc/</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">hf_qc/</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">|-- hf_d.manifest</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-quote">`-- qc.pbs</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><p><span>Check the commands generated </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ cat</span> hf_qc/qc.pbs</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-meta">#!/bin/bash</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#PBS -l nodes=1:ppn=1</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#PBS -l mem=80gb</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#PBS -l walltime=100:00:00</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-keyword">export</span> <span class="cm-def">LC_ALL</span><span class="cm-operator">=</span>en_AU.utf8</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-keyword">export</span> <span class="cm-def">LANG</span><span class="cm-operator">=</span>en_AU.utf8</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cd</span> /srv/scratch/z3524677/hfdiet/demo/hf_qc</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">' fastp -i all_r1.fq -I all_r2.fq -o /srv/scratch/z3524677/hfdiet/demo/hf_qc/ALL_R1.fq -O /srv/scratch/z3524677/hfdiet/demo/hf_qc/ALL_R2.fq -h /srv/scratch/z3524677/hfdiet/demo/hf_qc/fastp.outreport.html'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && qiime tools import --type '</span>SampleData[PairedEndSequencesWithQuality]<span class="cm-string">' --input-path /srv/scratch/z3524677/hfdiet/demo/hf_d.manifest --output-path demux.qza --input-format PairedEndFastqManifestPhred33'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trunc-len-f 295 --p-trunc-len-r 220 --p-trim-left-f 17 --p-trim-left-r 21 --p-n-threads 1 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats stats-dada2.qza'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 462px;"></div><div class="CodeMirror-gutters" style="display: none; height: 462px;"></div></div></div></pre><p><span>Files in the folder after running the </span><strong><em><span>mima_prepare_manifest_and_qc.pl</span></em></strong><span> script. Now a file 'hf_d.manifest' and a folder 'hf_qc' are newly created.</span></p><p><img src="./images/image-20200914124519856.png" referrerpolicy="no-referrer" alt="image-20200914124519856"></p><h2><a name="overall-assessment-of-quality-of-sequences-with-fastp" class="md-header-anchor"></a><span>Overall assessment of quality of sequences with fastp </span></h2><p><span>Prior to importing data into QIIME, the data will be assessed with Fastp. This step functions to assess overall sequence quality. Output information will be in a html format and will include: Run </span><a href='https://github.com/OpenGene/fastp'><span>Fastp</span></a><span> to assess the overall quality of the sequences. </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'fastp -i all_r1.fq -I all_r2.fq -o hf_qc/ALL_R1.fq -O hf_qc/ALL_R2.fq -h hf_qc/fastp.outreport.html'</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">bash</span>: warning: setlocale: LC_ALL: cannot change locale (en_AU.utf8)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Read1 before filtering:</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total reads: <span class="cm-number">40000</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total bases: <span class="cm-number">12022031</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q20 bases: <span class="cm-number">10726471</span>(89.2235%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q30 bases: <span class="cm-number">9355072</span>(77.8161%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Read2 before filtering:</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total reads: <span class="cm-number">40000</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total bases: <span class="cm-number">12001486</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q20 bases: <span class="cm-number">9746062</span>(81.2071%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q30 bases: <span class="cm-number">8306044</span>(69.2085%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Read1 after filtering:</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total reads: <span class="cm-number">38912</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total bases: <span class="cm-number">11695532</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q20 bases: <span class="cm-number">10522964</span>(89.9742%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q30 bases: <span class="cm-number">9202096</span>(78.6804%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Read2 aftering filtering:</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total reads: <span class="cm-number">38912</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total bases: <span class="cm-number">11674221</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q20 bases: <span class="cm-number">9568399</span>(81.9618%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Q30 bases: <span class="cm-number">8182633</span>(70.0915%)</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Filtering result:</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">reads passed filter: <span class="cm-number">77824</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">reads failed due to low quality: <span class="cm-number">1952</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">reads failed due to too many N: <span class="cm-number">224</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">reads failed due to too short: <span class="cm-number">0</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">reads with adapter trimmed: <span class="cm-number">24</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">bases trimmed due to adapters: <span class="cm-number">1575</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Duplication rate: <span class="cm-number">22</span>.3048%</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Insert size peak (evaluated by paired-end reads): <span class="cm-number">460</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">JSON report: fastp.json</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">HTML report: /srv/scratch/z3524677/hfdiet/demo/hf_qc/fastp.outreport.html</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 902px;"></div><div class="CodeMirror-gutters" style="display: none; height: 902px;"></div></div></div></pre><p><span>Check the output directory </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ ls</span> <span class="cm-attribute">-lh</span> hf_qc/ </span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">total 51M</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-attribute">-rw-r--r--</span>. <span class="cm-number">1</span> z3524677 unsw 26M Sep <span class="cm-number">9</span> <span class="cm-number">12</span>:09 ALL_R1.fq</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-attribute">-rw-r--r--</span>. <span class="cm-number">1</span> z3524677 unsw 26M Sep <span class="cm-number">9</span> <span class="cm-number">12</span>:09 ALL_R2.fq</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-attribute">-rw-r--r--</span>. <span class="cm-number">1</span> z3524677 unsw 436K Sep <span class="cm-number">9</span> <span class="cm-number">12</span>:09 fastp.outreport.html</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-attribute">-rw-r--r--</span>. <span class="cm-number">1</span> z3524677 unsw <span class="cm-number">2</span>.9K Sep <span class="cm-number">9</span> <span class="cm-number">12</span>:02 hf_d.manifest</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-attribute">-rw-r--r--</span>. <span class="cm-number">1</span> z3524677 unsw <span class="cm-number">1</span>.1K Sep <span class="cm-number">9</span> <span class="cm-number">12</span>:02 qc.pbs</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><span>Fastp Output explained:</span></p><ul><li><span>Insert size is the length of DNA that you want to sequence and that is inserted between the adapters (adapters excluded).</span></li><li><span>Total reads refer to the total number of reads generated by the sequencing run</span></li><li><span>Total bases refer to the total number of nucleotide bases sequenced during the sequencing run </span></li><li><span>Quality Scores for Next-Generation Sequencing (NGS) assessing sequencing accuracy using Phred quality scoring (Q score). Low Q scores can increase false-positive variant calls, which can result in inaccurate conclusions and higher costs for validation experiments.</span></li></ul><figure><table><thead><tr><th><span>Phred Quality</span></th><th><span>Score</span><span> </span><span>Probability of incorrect bases call</span></th><th><span>Base call accuracy</span></th></tr></thead><tbody><tr><td><span>10</span></td><td><span>1 in 10</span></td><td><span>90%</span></td></tr><tr><td><span>20</span></td><td><span>1 in 100</span></td><td><span>99%</span></td></tr><tr><td><span>30</span></td><td><span>1 in 1000</span></td><td><span>99.9%</span></td></tr></tbody></table></figure><p><span>Table: Phred Quality table</span></p><p><span> Check the quality of the overall sequences in the </span><strong><em><span>fastp.outreport.html</span></em></strong><span> after downloading to local computer</span></p><p><strong><em><span>fastp.outreport.html</span></em></strong><span> open on local browser</span></p><p><img src="./images/image-20200914123316926.png" alt="image-20200914123316926" style="zoom:90%;" /></p><p><strong><span>Determine the trim location at read1 and read2</span></strong>
<span>For Miseq reads, usually the quality of bases decrease dramatically while towarding to the end of the reads, in order to loss less sequences during merge of pair end reads, it is better operation to trim the end of reads.</span></p><p><span>Read1, to keep higher quality bases, the trim location is set to 295 </span>
<img src="./images/read1.png" alt="image-read1" style="zoom:90%;" /></p><p><span>Read2, to keep higher quality bases, the trim locaiton is set to 220</span><br/><img src="./images/read2.png" alt="image-read2" style="zoom:90%;" /></p><h2><a name="import-fasta-sequences-into-demuxqza" class="md-header-anchor"></a><span>Import fasta sequences into demux.qza </span></h2><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools import \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --type '</span>SampleData[PairedEndSequencesWithQuality]<span class="cm-string">' \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_d.manifest \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path demux.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-format PairedEndFastqManifestPhred33'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Imported /srv/scratch/z3524677/hfdiet/demo/hf_d.manifest as PairedEndFastqManifestPhred33 to demux.qza</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 198px;"></div><div class="CodeMirror-gutters" style="display: none; height: 198px;"></div></div></div></pre><p><span>Pay attention to the PATH of the input and output files in the command, make sure it is your PATH, e.g., /srv/scratch/'yourzID'/hfdiet/demo/hf_d.manifest. A file 'demux.qza' is newly generated.</span></p><p><img src="./images/image-20200914125411543.png" alt="image-20200914125411543" style="zoom:90%;" /></p><h2><a name="running-dada2-within-qiime2-to-perform-quality-control-this-takes-about-1-minute" class="md-header-anchor"></a><span>Running dada2 within qiime2 to perform quality control, this takes about 1 minute</span></h2><p><span>Run dada2 to trim end low quality bases, remove chimera sequencing and merge pair-end sequences, generate feature tables and statistics output of the results </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> [z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> qiime dada2 denoise-paired \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-demultiplexed-seqs demux.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-trunc-len-f 295 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-trunc-len-r 220 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-trim-left-f 17 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-trim-left-r 21 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-n-threads 1 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-representative-sequences rep-seqs.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-table table.qza --o-denoising-stats stats-dada2.qza'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> Saved FeatureTable[Frequency] to: table.qza</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> Saved FeatureData[Sequence] to: rep-seqs.qza</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"> Saved SampleData[DADA2Stats] to: stats-dada2.qza</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 330px;"></div><div class="CodeMirror-gutters" style="display: none; height: 330px;"></div></div></div></pre><p><span>Three files: 'table.qza', 'rep-seqs.qza' and 'stats-dada2.qza' are newly generated.</span></p><p><img src="./images/image-20200914130427694.png" alt="image-20200914130427694" style="zoom:80%;" /></p><p> </p><p><span>Decompress </span><strong><span>stats-dada2.qza</span></strong><span> and check the quality details: </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path stats-dada2.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path stats-dada2'</span> </span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><p><strong><span>Dada2 stats</span></strong></p><ul><li><span>The </span><strong><span>input column</span></strong><span> refers to the sequence reads imported into QIIME2 from the initial QC filtration. </span></li><li><span>Sequence reads are then filtered/denoised using parameters set by user. These are determined based on sequence 16SrRNA gene region amplified and quality assessment from the fastp report.</span></li><li><span>The </span><strong><span>merge column</span></strong><span> (only present for paired end samples) refers to the number of the forward and reverse reads which were successfully merged together to obtain the full denoised sequences. Merging is performed by aligning the denoised forward reads with the reverse-complement of the corresponding denoised reverse reads, and then constructing the merged “contig” sequences. By default, merged sequences are only output if the forward and reverse reads overlap by at least 12 bases, and are identical to each other in the overlap region.</span></li><li><span>The </span><strong><span>non-chimeric column</span></strong><span> refers to the number of sequences remaining after chimera removal. Chimeras are artifactual PCR product/amplicon generated erroneously from more than one DNA template. It is well-known that chimeras are inevitable when preparing amplicon sequencing libraries for NGS. It is therefore important to detect and filter them out before any types of microbiome analyses. </span></li></ul><p><span>Look into the quality details:</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cat</span> stats-dada2/stats.tsv</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output:</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">sample-id<span class="cm-tab" role="presentation" cm-text=" "> </span>input<span class="cm-tab" role="presentation" cm-text=" "> </span>filtered<span class="cm-tab" role="presentation" cm-text=" "> </span>percentage of input passed filter<span class="cm-tab" role="presentation" cm-text=" "> </span>denoised<span class="cm-tab" role="presentation" cm-text=" "> </span>merged<span class="cm-tab" role="presentation" cm-text=" "> </span>percentage of input merged<span class="cm-tab" role="presentation" cm-text=" "> </span>non-chimeric<span class="cm-tab" role="presentation" cm-text=" "> </span>percentage of input non-chimeric</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-comment">#q2:types<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric<span class="cm-tab" role="presentation" cm-text=" "> </span>numeric</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5821_S50_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1337</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">53</span>.48<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1215</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1089</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">43</span>.56<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1073</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">42</span>.92</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5822_S34_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1467</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">58</span>.68<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1364</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1256</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">50</span>.24<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1243</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">49</span>.72</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5824_S96_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1312</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">52</span>.48<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1203</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1100</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">44</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1098</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">43</span>.92</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5825_S7_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1365</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">54</span>.6<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1268</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1131</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">45</span>.24<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1116</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">44</span>.64</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5832_S8_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1350</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">54</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1229</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1069</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">42</span>.76<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1058</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">42</span>.32</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5833_S78_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1218</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">48</span>.72<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1092</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">881</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">35</span>.24<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">877</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">35</span>.08</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5859_S27_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1279</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">51</span>.16<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1138</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">994</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">39</span>.76<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">987</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">39</span>.48</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5860_S122_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1449</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">57</span>.96<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1344</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1229</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">49</span>.16<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1225</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">49</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5862_S51_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1232</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">49</span>.28<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1121</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1000</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">40</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">998</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">39</span>.92</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5863_S30_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1371</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">54</span>.84<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1263</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1140</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">45</span>.6<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1133</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">45</span>.32</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6319_S128_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1248</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">49</span>.92<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1176</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1025</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">41</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">987</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">39</span>.48</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6320_S67_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1283</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">51</span>.32<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1180</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">985</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">39</span>.4<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">951</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">38</span>.04</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6321_S20_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1339</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">53</span>.56<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1190</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">973</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">38</span>.92<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">932</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">37</span>.28</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6322_S131_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1466</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">58</span>.64<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1339</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1116</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">44</span>.64<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1045</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">41</span>.8</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6328_S93_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1531</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">61</span>.24<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1420</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1256</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">50</span>.24<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1146</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">45</span>.84</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">6329_S37_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">2500</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1327</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">53</span>.08<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1217</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1012</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">40</span>.48<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">1000</span><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">40</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 506px;"></div><div class="CodeMirror-gutters" style="display: none; height: 506px;"></div></div></div></pre><p><span>**Determine the depth to normalize for alpha and beta diversity comparison</span>
<span>By analyzing the depth of clean sequences of all the samples, 800 is decided as the cut off for normalization to keep all samples. </span></p><p><span>Contents of two important files </span><strong><span>rep-seqs.qza</span></strong><span> and </span><strong><span>table.qza</span></strong></p><p><strong><span>rep-seqs.qza</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path rep-seqs.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path rep-seqs'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#Exported rep-seqs.qza as DNASequencesDirectoryFormat to directory rep-seqs</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>Look the feature sequences </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">head <span class="cm-attribute">-4</span> rep-seqs/dna-sequences.fasta</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output: </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">>dfa833b266bd2993b86feab3617b34c3</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TCGAGAATCATTCACAATGGGGGAAACCCTGATGGTGCGACGCCGCGTGGGGGAATGAAGGTCTTCGGATTGTAAACCCCTGTCATGTGGGAGCAAATTAAAAAGATAGTACCACAAGAGGAAGAGACGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGTCTCAAGCGTTGTTCGGAATCACTGGGCGTAAAGCGTGCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAGGCGCGGGCTCAACCCGCGGACGGCACATGATACTGCGAGACTAGAGTAATGGAGGGGGAACCGGAATTCTCGGTGTAGCAGTGAAATGCGTAGATATCGAGAGGAACACTCGTGGCGAAGGCGGGTTCCTGGACATTAACTGACGCTGAGGCACGAAGGCCAGGGGAGCGAAAG</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">>f67a6d5da8c0b71206955aeae1505a74</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TGAGGAATATTGGTCAATGGTCGGGAGACTGAACCAGCCAAGCCGCGTGAGGGAAGAAGGTACAGCGTATCGTAAACCTCTTTTGCCGGGGAACAAGAGCTTCCACGAGTGGGAGTGTGAGCGTACCCGGAGAAAAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCGAGTTAAGTCAGCGGTAAAAGCCCGGGGCTCAACCCCGGCCCGCCGTTGAAACTGGCTGGCTTGAGTTGGGGAAAGGCAGGCGGAATGCGCGGTGTAGCGGTGAAATGCATAGATATCGCGCAGAACCCCGATTGCGAAGGCAGCCTGCCGGCCCCACACTGACGCTGAGGCACGAAAGCGTGGGTATCGAACA</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 286px;"></div><div class="CodeMirror-gutters" style="display: none; height: 286px;"></div></div></div></pre><p><strong><span>table.qza</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path table.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path table'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#Exported table.qza as BIOMV210DirFmt to directory table</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#convert biom file to tsv file </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">biom convert -i table/feature-table.biom -o table/feature-table.txt --to-tsv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 198px;"></div><div class="CodeMirror-gutters" style="display: none; height: 198px;"></div></div></div></pre><p><span>Look into the table matrix file, the numbers in the table are abundance of that feature sequences in different samples</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">head <span class="cm-attribute">-4</span> table/feature-table.txt</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output:</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment"># Constructed from biom file</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-comment">#OTU ID<span class="cm-tab" role="presentation" cm-text=" "> </span>5821_S50_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5822_S34_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5824_S96_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5825_S7_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5832_S8_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5833_S78_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5859_S27_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5860_S122_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5862_S51_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>5863_S30_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>6319_S128_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>6320_S67_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>6321_S20_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>6322_S131_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>6328_S93_L001<span class="cm-tab" role="presentation" cm-text=" "> </span>6329_S37_L001</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">dfa833b266bd2993b86feab3617b34c3<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">193</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">186</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">271</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">290</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">208</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">176</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">210</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">322</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">223</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">230</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">140</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">63</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">54</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">117</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">0</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">0</span>.0</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">f67a6d5da8c0b71206955aeae1505a74<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">55</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">126</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">72</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">85</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">57</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">39</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">46</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">51</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">48</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">36</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">131</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">35</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">38</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">0</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">109</span>.0<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">14</span>.0</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 264px;"></div><div class="CodeMirror-gutters" style="display: none; height: 264px;"></div></div></div></pre><p> </p><h2><a name="host-contamination-removal-if-necessary-when-processing-mucosal-samples" class="md-header-anchor"></a><span>Host contamination removal if necessary when processing mucosal samples</span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand why there are contamination in the amplicons data</span></li><li><span>Finish the commands to decontamination</span></li></ul><p><span>Although the sequencing target is the 16S hyper variable region PCR products, the data generated by Ramaciotti sometimes includes host contaminations. Hence, we need to remove the host sequences in the </span><strong><span>table.qza</span></strong><span> and </span><strong><span>rep-seq.qza</span></strong><span>, and update the stats-dada2.qza again to check the percentage of host contamination. </span></p><p><span>Check the help information for </span><strong><span>mima_decontaminate_host.pl</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && perl /home/applications/mima/mima_decontaminate_host.pl '</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">perl /home/applications/mima/mima_decontaminate_host.pl <rep_seqs.qza> <table.qza> <outputdir> <host_genome.fa_bowtie2_index> <threads></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><rep_seqs.qza> qiime format represntative sequences output from dada2</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><table.qza> qimme format features table</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><outputdir> output directory to store the decontaminated updated rep_seqs.qza and table.qza</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><host_genome.fa_bowtie2_index> bowtie2 index of the host genome</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><threads> number of threads used <span class="cm-keyword">in</span> bowtie2 mapping</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span>This script use bowtie2 to maping the rep_seqs.qza aganist host genome to identify the contaminated sequences and update the sequences and table with qiime2. The updated files can be used <span class="cm-keyword">for</span> downstream analysis.</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 286px;"></div><div class="CodeMirror-gutters" style="display: none; height: 286px;"></div></div></div></pre><p><span>Run the decontamination script to generate the bash file and run the analysis with singularity</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ singularity</span> exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && perl /home/applications/mima/mima_decontaminate_host.pl rep-seqs.qza table.qza hf_host_decontamination /data/bio/workshop/db/mouse.genome 1'</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">[z3524677@katana2 demo]<span class="cm-def">$ ls</span> hf_host_decontamination/</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">decontaminationhost.sh</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>A folder 'hf_host_decontamination/' is newly creatd, with one file 'decontaminationhost.sh' in it.</span></p><p><img src="./images/image-20200914133031716.png" alt="image-20200914133031716" style="zoom:80%;" /></p><p><strong><span>Execute the shell commands</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && sh hf_host_decontamination/decontaminationhost.sh'</span></span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 44px;"></div><div class="CodeMirror-gutters" style="display: none; height: 44px;"></div></div></div></pre><p><span>Aftering running the decontamination pipeline, we get an updated </span><strong><span>rep-seqs.qza</span></strong><span> and </span><strong><span>table.qza</span></strong></p><p><strong><span>Explain of the output folder</span></strong></p><ul><li><span>The file 'align-hostgenome.sam.txt' in folder 'rep-seqsold' holds the mapping info of the fasta sequences in 'dna-sequences.fasta'. Due to the small size of the demo data, there is no host reads detected therefore no record in 'align-hostgenome.sam.txt'.</span></li><li><span>The file 'metadata.table.txt' holds the IDs of the fasta sequences that are mapped to host reference, therefore there is no record in this file, either.</span></li><li><span>The file 'filtered_dnaseqs.fasta' has the fasta sequences that are not mapped to host reference, therefore we shall focus on this file in the downstream analysis.</span></li></ul><p><img src="./images/image-20200914133227730.png" referrerpolicy="no-referrer" alt="image-20200914133227730"></p><h1><a name="3-taxonomy-annotation-and-diversity-analysis" class="md-header-anchor"></a><span>3. Taxonomy annotation and diversity analysis </span></h1><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 60 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Running diversity analysis pipeline and taxonomy annotation, alpha and beta diversity analysis </span></li><li><span>Understand how to interpret the output </span></li></ul><p><span>Taxonomy annotation of </span><strong><span>rep-seqs.qza</span></strong><span> with naive bayesian classifier, for this part analysis, we have a comprehensive pipeline script, it can generate a series of commands that can finish different analysis, we firstly run this pipeline and then go to details of each part of the flow. </span></p><p> </p><p><span>Create a directory </span><strong><span>hf_diver</span></strong><span> under the demo directory and copy the updated rep-seqs.qza and table.qza into this folder </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#make the diversity output directory</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">mkdir</span> hf_diver</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#move the two files feature table and featues sequences into the hf_diver directory </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cp</span> rep-seqs.qza hf_diver/</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-builtin">cp</span> table.qza hf_diver/</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>Execute taxonomy annotation and diversity analysis pipeline command </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">perl /home/applications/mima/mima_diversity-basic-statistic.pl \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> -m meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> -c /data/bio/workshop/db/gg-v3v4-classifier.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> -n 1 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> -d 800 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> -o hf_diver'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><span>The </span><strong><span>mama_diversity-basic-statistic.pl</span></strong><span> pipeline will generate a shell file </span><em><span>beta-diversity-commands.sh</span></em><span>, which includes all the taxonomical, alpha, beta diversity analysis . A file </span><em><span>beta-diversity-commands.sh</span></em><span> and a folder </span><strong><span>hf_diver</span></strong><span> are newly generated. </span></p><p><img src="./images/image-20200914134531091.png" alt="image-20200914134531091" style="zoom:80%;" /></p><p><strong><span>Run the above diversity analysis in batch model</span></strong><span> (This command run the pipeline)</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#Run the analysis in batch model, the /scratch should be bind for this function, the database directory should be bind as well to get access to classifier database </span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec <span class="cm-attribute">-B</span> /data/bio/workshop/db/,/scratch /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && sh beta-diversity-commands.sh '</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><h2><a name="taxonomy-assignment-of-feature-sequences" class="md-header-anchor"></a><span>Taxonomy assignment of feature sequences </span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand what is a phylogenetic tree and the purpuse of why we need this</span></li><li><span>Finish the commands to generate rooted tree</span></li></ul><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#taxonomy assignment of feature representative sequences</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec <span class="cm-attribute">-B</span> /data/bio/workshop/db/ /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime feature-classifier classify-sklearn \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-classifier /data/bio/workshop/db/gg-v3v4-classifier.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-reads hf_diver/rep-seqs.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-classification hf_diver/taxonomy.qza'</span> </span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><strong><span>Check the contents of the taxonomy.qza by decompress it and view it</span></strong></p><p><span>Decompress </span><em><span>hf_diver/taxonomy.qza</span></em></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_diver/taxonomy.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path hf_diver/taxonomy'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output meassage </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#Exported hf_diver/taxonomy.qza as TSVTaxonomyDirectoryFormat to directory hf_diver/taxonomy</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><span>Check the taxonomic annotaion of represent sequences </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">head <span class="cm-attribute">-4</span> hf_diver/taxonomy/taxonomy.tsv</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">Feature ID<span class="cm-tab" role="presentation" cm-text=" "> </span>Taxon<span class="cm-tab" role="presentation" cm-text=" "> </span>Confidence</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">dfa833b266bd2993b86feab3617b34c3<span class="cm-tab" role="presentation" cm-text=" "> </span>k__Bacteria; p__Verrucomicrobia; c__Verrucomicrobiae; o__Verrucomicrobiales; f__Verrucomicrobiaceae; g__Akkermansia; s__muciniphila<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">0</span>.9999999999999716</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">f67a6d5da8c0b71206955aeae1505a74<span class="cm-tab" role="presentation" cm-text=" "> </span>k__Bacteria; p__Bacteroidetes; c__Bacteroidia; o__Bacteroidales; f__S24-7; g__; s__<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">0</span>.9999839589828744</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">d272bf25781448dde9031a24679a9012<span class="cm-tab" role="presentation" cm-text=" "> </span>k__Bacteria; p__Bacteroidetes; c__Bacteroidia; o__Bacteroidales; f__Bacteroidaceae; g__Bacteroides; s__acidifaciens<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">0</span>.9995083080422182</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 220px;"></div><div class="CodeMirror-gutters" style="display: none; height: 220px;"></div></div></div></pre><p><strong><span>Normalize the feature table to a uniform depth</span></strong></p><p><strong><span>Note:</span></strong><span> The decision of how deep should be selected is based on </span><strong><span>stats-dada2/stats.tsv</span></strong><span> clean reads in all samples, you should keep as much samples as possible, and should keep a good enough depth, usually for stool samples over 10000 sequences should be fine. Here, as this is a demo, the raw sequences are 2500 and the final clean reads are all over 800, we chose 800 reads as the cut off. </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime feature-table rarefy \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-table hf_diver/table.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-sampling-depth 800 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-rarefied-table hf_diver/rarefy.table.qza'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><strong><span>Generate stack barchart</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime taxa barplot \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-table hf_diver/rarefy.table.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-taxonomy hf_diver/taxonomy.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/normalized.taxa-bar-plots.qzv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><strong><span>Decompress qzv file and download to local to browse the file</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_diver/normalized.taxa-bar-plots.qzv \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path hf_diver/normalized.taxa-bar-plots'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><p><span>Three files 'taxonomy.qza', 'rarefy.table.qza', 'normalized.taxa-bar-plots.qzv' and one filder 'normalized.taxa-bar-plots' are generated sequentially.</span></p><p><img src="./images/image-20200914135959510.png" alt="image-20200914135959510" style="zoom:80%;" /></p><p><span>If you download file 'normalized.taxa-bar-plots.qzv' locally then upload to qiime2 view, the visualization should be look like this. 'Taxonomic Level', 'Color Palette' and 'Sort Samples By' all can be manully modified. </span></p><p><img src="./images/image-barplot.png" alt="image-20200914140443087" style="zoom:80%;" /></p><h2><a name="phylogenetic-tree-construction" class="md-header-anchor"></a><span>Phylogenetic tree construction </span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand what is a phylogenetic tree and the purpuse of why we need this</span></li><li><span>Finish the commands to generate rooted tree</span></li></ul><p><span>In order to generate the UniFrac distance, a phylogenetic tree should be build for all the representative feature sequences</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#constructing phylogenetic tree</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec <span class="cm-attribute">-B</span> /scratch /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime alignment mafft \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-sequences hf_diver/rep-seqs.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-alignment hf_diver/aligned-rep-seqs.qza'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-tab" role="presentation" cm-text=" "> </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec <span class="cm-attribute">-B</span> /scratch /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime alignment mask \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-alignment hf_diver/aligned-rep-seqs.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-masked-alignment hf_diver/masked-aligned-rep-seqs.qza'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-tab" role="presentation" cm-text=" "> </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec <span class="cm-attribute">-B</span> /scratch /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime phylogeny fasttree \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-alignment hf_diver/masked-aligned-rep-seqs.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-tree hf_diver/unrooted-tree.qza --p-n-threads 1'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec <span class="cm-attribute">-B</span> /scratch /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime phylogeny midpoint-root \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-tree hf_diver/unrooted-tree.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-rooted-tree hf_diver/rooted-tree.qza'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 440px;"></div><div class="CodeMirror-gutters" style="display: none; height: 440px;"></div></div></div></pre><p><span>Four files 'aligned-rep-seqs.qza', 'masked-aligned-rep-seqs.qza', 'unrooted-tree.qza' and 'rooted-tree.qza' are generated sequentially. The </span><strong><span>rooted-tree.qza</span></strong><span> will be used in the core diversity analysis </span></p><p><img src="./images/image-20200914141104790.png" alt="image-20200914141104790" style="zoom:80%;" /></p><h2><a name="run-the-core-diversity-analysis" class="md-header-anchor"></a><span>Run the core diversity analysis</span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand the output for the core diversity analysis </span></li><li><span>Checking the results for PcoA with different beta diversity distance</span></li></ul><p><span>Sometimes we need to filter samples by the meta data to select part of the samples for analysis</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime feature-table filter-samples \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-table hf_diver/table.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-filtered-table hf_diver/core.table.qza'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><strong><span>core diversity with phylogenetic information</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity core-metrics-phylogenetic \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-phylogeny hf_diver/rooted-tree.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-table hf_diver/core.table.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-sampling-depth 800 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-dir hf_diver/core-metrics-results'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><span>One files 'core.table.qza' and one filder 'core-metrics-results', which has the core diversity analysis outputs, are generated sequentially. This step generate alpha diversity indexes such a Shannon, observed features, jaccard, evenness, and faith phylogenetic distance data and plot. The beta diversity of PcoA such as bray-curtis distance, weighted and unweighted UniFrac distance. </span></p><p><img src="./images/image-20200914141242714.png" alt="image-20200914141242714" style="zoom:80%;" /><span>d</span></p><p> </p><p><strong><span>Check alpha diversity contents, shannon vector for example</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_diver/core-metrics-results/shannon_vector.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path hf_diver/core-metrics-results/shannon_vector'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><p><span>Look into the output file</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">head <span class="cm-attribute">-4</span> hf_diver/core-metrics-results/shannon_vector/alpha-diversity.tsv</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-comment">#output: </span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span>shannon_entropy</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5821_S50_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">4</span>.284775238361043</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5822_S34_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">4</span>.492180841533424</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">5824_S96_L001<span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-number">4</span>.474363582402979</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><strong><span>Check PcoA plot for bray-curtis distance and weighted unifrac distance</span></strong></p><p><span>Decompress the qzv file and visulize at local computer or donwload qzv file and upload to qiime2 view for visulization </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_diver/core-metrics-results/weighted_unifrac_emperor.qzv \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path hf_diver/core-metrics-results/weighted_unifrac_emperor'</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><span>If you download file 'bray_curtis_emperor.qzv' locally then upload to </span><a href='https://view.qiime2.org/'><span>qiime2view</span></a><span>, the visualization should be look like this. (Red is high fat diet samples and blue are healthy)</span></p><p><img src=".\images\emperor.png" alt="emperor" style="zoom:60%;" /></p><p><span>If you download file 'weighted_unifrac_emperor.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/emperor.3.png" referrerpolicy="no-referrer" alt="emperor (3)"></p><p><strong><span>Now check all the other alpha diversity index and beta diveristy PcoA results 5 minutes given</span></strong></p><h2><a name="alpha-diversity-rarefaction-curve" class="md-header-anchor"></a><span>Alpha diversity rarefaction curve</span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 5 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand the meaning of rarefaction curve</span></li><li><span>Generating rarefaction curve with qiime diversity plugin</span></li></ul><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity alpha-rarefaction \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-table hf_diver/table.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-phylogeny hf_diver/rooted-tree.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --p-max-depth 800 \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/alpha-rarefaction.qzv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 154px;"></div><div class="CodeMirror-gutters" style="display: none; height: 154px;"></div></div></div></pre><p><span>A file 'alpha-rarefaction.qzv' is created.</span></p><p><img src="./images/image-20200914142559123.png" alt="image-20200914142559123" style="zoom:80%;" /></p><p><span>If you download file 'alpha-rarefaction.qzv' locally then upload to qiime2 view, the visualization should be look like this. 'Metric' has options (1) observed_features, (2) shannon and (3) faith_pd.</span></p><p><img src="./images/image-20200913173239270.png" alt="image-20200913173239270" style="zoom:50%;" /></p><h2><a name="permanova-statistical-testing-of-alpha-and-beta-diversity-with-meta-data" class="md-header-anchor"></a><span>PERMANOVA statistical testing of alpha and beta diversity with meta data</span></h2><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 10 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand PERMANONA testing for group significance</span></li><li><span>Identifying the most significant influential meta data that associated with microbial alpha and beta diversity </span></li></ul><h3><a name="permanova-statistical-testing-of-alpha-diversity-for-groups" class="md-header-anchor"></a><span>PERMANOVA statistical testing of alpha diversity for groups</span></h3><p><span>Faith_ph</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity alpha-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-alpha-diversity hf_diver/core-metrics-results/faith_pd_vector.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/alpha-group/faith-pd-group-significance.qzv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><span>A file 'faith-pd-group-significance.qzv' is newly generated.</span></p><p><img src="./images/image-20200914142935737.png" alt="image-20200914142935737" style="zoom:80%;" /></p><p><span>If you download file 'faith-pd-group-significance.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913173710256.png" alt="image-20200913173710256" style="zoom:50%;" /></p><p><span>evenness</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity alpha-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-alpha-diversity hf_diver/core-metrics-results/evenness_vector.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/alpha-group/evenness-group-significance.qzv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><span>A file 'evenness-group-significance.qzv' is newly generated.</span></p><p><img src="./images/image-20200914143441377.png" alt="image-20200914143441377" style="zoom:80%;" /></p><p><span>If you download file 'evenness-group-significance.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913173749861.png" alt="image-20200913173749861" style="zoom:50%;" /></p><p><span>observed features </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity alpha-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-alpha-diversity hf_diver/core-metrics-results/observed_features_vector.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/alpha-group/observed_features_vector.qzv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><span>A file 'observed_features_vector.qzv' is newly generated.</span></p><p><img src="./images/image-20200914143559083.png" alt="image-20200914143559083" style="zoom:80%;" /></p><p><span>If you download file 'observed_features_vector.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913173824880.png" alt="image-20200913173824880" style="zoom:50%;" /></p><p><span>shannon index </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity alpha-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-alpha-diversity hf_diver/core-metrics-results/shannon_vector.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/alpha-group/shannon_vector.qzv'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 110px;"></div><div class="CodeMirror-gutters" style="display: none; height: 110px;"></div></div></div></pre><p><span>A file 'shannon_vector.qzv' is newly generated.</span></p><p><img src="./images/image-20200914143658998.png" referrerpolicy="no-referrer" alt="image-20200914143658998"></p><p><span>If you download file 'shannon_vector.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913173852379.png" alt="image-20200913173852379" style="zoom:50%;" /></p><p><span>Output alpha diversity values from qiime2 output</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_diver/alpha-group/shannon_vector.qzv \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path hf_diver/alpha-group/shannon_vector'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><p><span>A folder 'shannon_vector' is newly generated. You can download the whole directory to your local pc or laptap and open the file 'index.html' to visualize.</span></p><p><img src="./images/image-20200914143913015.png" referrerpolicy="no-referrer" alt="image-20200914143913015"></p><h3><a name="permanova-statistical-testing-of-beta-diversity-for-groups" class="md-header-anchor"></a><span>PERMANOVA statistical testing of beta diversity for groups </span></h3><p><span>Bray curtis distance on diet</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity beta-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-distance-matrix hf_diver/core-metrics-results/bray_curtis_distance_matrix.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-column "Diet" \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/beta-group/Diet.bray_curtis.qzv --p-pairwise'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>A file 'Diet.bray_curtis.qzv' is newly generated.</span></p><p><img src="./images/image-20200914144118726.png" alt="image-20200914144118726" style="zoom:100%;" /></p><p><span>If you download file 'Diet.bray_curtis.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913174248563.png" alt="image-20200913174248563" style="zoom:50%;" /></p><p><span>Jaccard distance </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity beta-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-distance-matrix hf_diver/core-metrics-results/jaccard_distance_matrix.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-column "Diet" \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/beta-group/Diet.jaccard.qzv --p-pairwise'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>A file 'Diet.jaccard.qzv' is newly generated.</span></p><p><img src="./images/image-20200914144420279.png" referrerpolicy="no-referrer" alt="image-20200914144420279"></p><p><span>If you download file 'Diet.jaccard.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913174321071.png" alt="image-20200913174321071" style="zoom:50%;" /></p><p><span>Unweighted UniFrac distacne</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity beta-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-distance-matrix hf_diver/core-metrics-results/unweighted_unifrac_distance_matrix.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-column "Diet" \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/beta-group/Diet.unweighted_unifrac.qzv --p-pairwise'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>A file 'Diet.unweighted_unifrac.qzv' is newly generated.</span></p><p><img src="./images/image-20200914144522004.png" referrerpolicy="no-referrer" alt="image-20200914144522004"></p><p><span>If you download file 'Diet.unweighted_unifrac.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913174400910-1599984477027.png" alt="image-20200913174400910" style="zoom:50%;" /></p><p><span>Weighted UniFrac distacne </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime diversity beta-group-significance \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --i-distance-matrix hf_diver/core-metrics-results/weighted_unifrac_distance_matrix.qza \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-file meta_data.txt \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --m-metadata-column "Diet" \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --o-visualization hf_diver/beta-group/Diet.weighted_unifrac.qzv --p-pairwise'</span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 132px;"></div><div class="CodeMirror-gutters" style="display: none; height: 132px;"></div></div></div></pre><p><span>A file 'Diet.weighted_unifrac.qzv' is newly generated.</span></p><p><img src="./images/image-20200914144626873.png" referrerpolicy="no-referrer" alt="image-20200914144626873"></p><p><span>If you download file 'Diet.weighted_unifrac.qzv' locally then upload to qiime2 view, the visualization should be look like this.</span></p><p><img src="./images/image-20200913174431036.png" alt="image-20200913174431036" style="zoom:50%;" /></p><p><span>Decompress visulization qzv file and download to local computer to check the results </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && \</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string">qiime tools export \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --input-path hf_diver/beta-group/Diet.weighted_unifrac.qzv \</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-string"> --output-path hf_diver/beta-group/Diet.weighted_unifrac'</span> </span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 88px;"></div><div class="CodeMirror-gutters" style="display: none; height: 88px;"></div></div></div></pre><p><span>A folder 'Diet.weighted_unifrac' is newly generated. You can download the whole directory to your local pc or laptap and open the file 'index.html' to visualize.</span></p><p><img src="./images/image-20200914144714685.png" referrerpolicy="no-referrer" alt="image-20200914144714685"></p><h1><a name="4-using-lefse-to-identify-differential-abundant-taxa" class="md-header-anchor"></a><span>4. Using LEfSe to identify differential abundant taxa</span></h1><blockquote><h4><a name="overview" class="md-header-anchor"></a><span>Overview</span></h4><p><strong><span>Teaching:</span></strong><span> 20 min</span>
<strong><span>Exercises:</span></strong><span> 0 min</span></p></blockquote><p><strong><span>Objectives</span></strong></p><ul><li><span>Understand how LEfSe identify microbial signatures</span></li><li><span>Identify differential abundant taxa in the high fat diet mouse </span></li></ul><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate qiime2-2020.8 && perl /home/applications/mima/mima_lefsetable_16s.pl hf_diver/rarefy.table.qza hf_diver/taxonomy.qza Diet meta_data.txt hf_lefse_tables '</span></span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 66px;"></div><div class="CodeMirror-gutters" style="display: none; height: 66px;"></div></div></div></pre><p><span>LEfSe outputs are in directory 'hf_lefse_tables', this step will generate input file of differnt ranks and merge all 7 ranks taxa into one file to be used in the following step to draw clade plot and bar plot of LDA scores. </span></p><p><span>For a regular tab seperated table to do LEfSe analysis, the following pipeline can be used </span>
<span>Check the help information for the pipeline </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate lefse-conda && perl /home/applications/mima/mima_lefse_pipeline.pl '</span></span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span cm-text=""></span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-i</span> table.tsv, the rarefied feature table used to <span class="cm-keyword">do</span> LEfSe</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-m</span> meta datafile, a table with rows as sample and column as differnt meta data</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-g</span> group vector should be column_name:group1,group2,group3 <span class="cm-comment">#the detail groups in one column to compare</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-o</span> output dir/ string</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-f</span> prefix to the output file, string format</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-d</span> LDA schore forcut off default <span class="cm-number">2</span></span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-x</span> width of figure default <span class="cm-number">8</span> inch</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-y</span> height of figure default <span class="cm-number">8</span> inch</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span><span class="cm-attribute">-h</span> print this help information</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" class="cm-tab-wrap-hack" style="padding-right: 0.1px;"><span class="cm-tab" role="presentation" cm-text=" "> </span></span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 286px;"></div><div class="CodeMirror-gutters" style="display: none; height: 286px;"></div></div></div></pre><p><span>Here in this example, we would like to identify the differential abundant taxa that enriched in high fat diet mouse. </span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="shell"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="shell"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 8px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">singularity exec /data/bio/workshop/mrcmicrobiome.sif <span class="cm-builtin">bash</span> <span class="cm-attribute">-c</span> <span class="cm-string">'. activate lefse-conda && perl /home/applications/mima/mima_lefse_pipeline.pl -i hf_lefse_tables/lefse_allrank/merge_taxonomy.csv -m meta_data.txt -g Diet:Normal,HFD -o hf_lefsenew -f dietlefse -d 3'</span></span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 66px;"></div><div class="CodeMirror-gutters" style="display: none; height: 66px;"></div></div></div></pre><h2><a name="cladoplot-presenting-of-signatures" class="md-header-anchor"></a><span>cladoplot presenting of signatures</span></h2><p><img src="./images/image-20200914203142279.png" referrerpolicy="no-referrer" alt="image-20200914203142279"></p><p> </p><h2><a name="lefse-signatures-barchart" class="md-header-anchor"></a><span>LEfSe signatures barchart</span></h2><p> </p><p><img src="./images/image-20200914210527233.png" alt="image-20200914210527233" style="zoom:80%;" /></p><p><span>Congratulations! </span></p><hr /><p><span>The copyright of qiime2 and LEfSe belongs to the developing team. The singularity version of pipeline is develped by MRC Bioinformatics team. </span></p><p> </p></div>
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