Fix bugs

Author: MaikTungsten

README.md

@@ -21,19 +21,28 @@ This pipeline makes use of the following tools:
 
 ### Installation
 
-Installation of conda environment from file:
+Installation of conda environments from file:
 
 ```bash
-conda env create -f /env/env_nextflow.yml
+conda env create -f /env/RNASEQ.yml
+conda env create -f /env/MULTIQC.yml
 ```
 
 or, when using mamba:
 
 ```bash
-mamba env create -f /env/env_nextflow.yml
+mamba env create -f /env/RNASEQ.yml
+mamba env create -f /env/MULTIQC.yml
 ```
 
 Also, check if Nextflow is available, otherwise install manually.
+Please check that all required packages have been successfully installed in the environments:
+- subread
+- hisat2
+- fastQC
+- cutadapt
+- samtools
+- multiQC (only in MULTIQC env)
 
 
 ### Input & usage
@@ -43,7 +52,6 @@ Input can be specified from command line or in /nf/conf/params.config.
 - hostGenome, phageGenome: provide absolute paths to full genomic fasta files.
 - hostGFF, phageGFF: provide absolute paths to full genomic feature annotation files.
 - pairedEnd: call, if dealing with paired-end RNA-seq data
-- dRNAseq: this flag will direct to dRNA-seq processing, but is not yet implemented
 - reads: absolute path to sreads in fastq format, can also be compressed as *.fastq.gz
 - outputDir: absolute path to output directory
 - adapter1, adapter2: by deafult Illumina TruSeq adapter sequences, which can be adjusted, if others used

env/MULTIQC.yml

@@ -4,88 +4,90 @@ channels:
 dependencies:
   - _libgcc_mutex=0.1=main
   - _openmp_mutex=5.1=1_gnu
-  - backports=1.1=pyhd3eb1b0_1
-  - backports.functools_lru_cache=1.6.4=pyhd3eb1b0_0
-  - backports_abc=0.5=py_1
-  - blas=1.0=mkl
   - bzip2=1.0.8=h5eee18b_6
-  - c-ares=1.19.1=h5eee18b_0
-  - ca-certificates=2024.12.31=h06a4308_0
-  - certifi=2020.6.20=pyhd3eb1b0_3
-  - click=7.1.2=pyhd3eb1b0_0
-  - curl=8.12.0=h251f7ec_0
-  - cycler=0.10.0=py27_0
-  - dbus=1.13.18=hb2f20db_0
-  - expat=2.6.4=h6a678d5_0
-  - fontconfig=2.14.1=h52c9d5c_1
-  - freetype=2.12.1=h4a9f257_0
-  - functools32=3.2.3.2=py27_1
-  - futures=3.3.0=py27_0
-  - gettext=0.21.0=hf68c758_0
-  - git=2.45.2=pl5340h9abc3c3_1
-  - glib=2.78.4=h6a678d5_0
-  - glib-tools=2.78.4=h6a678d5_0
-  - gst-plugins-base=1.14.1=h6a678d5_1
-  - gstreamer=1.14.1=h5eee18b_1
-  - icu=58.2=he6710b0_3
-  - intel-openmp=2023.1.0=hdb19cb5_46306
-  - jinja2=2.11.3=pyhd3eb1b0_0
-  - jpeg=9e=h5eee18b_3
-  - kiwisolver=1.1.0=py27he6710b0_0
-  - krb5=1.20.1=h143b758_1
-  - libcurl=8.12.0=hc9e6f67_0
-  - libedit=3.1.20230828=h5eee18b_0
-  - libev=4.33=h7f8727e_1
+  - ca-certificates=2025.11.4=h06a4308_0
+  - expat=2.7.3=h3385a95_0
+  - ld_impl_linux-64=2.44=h153f514_2
   - libffi=3.4.4=h6a678d5_1
-  - libgcc-ng=11.2.0=h1234567_1
-  - libgfortran-ng=7.5.0=ha8ba4b0_17
-  - libgfortran4=7.5.0=ha8ba4b0_17
-  - libglib=2.78.4=hdc74915_0
-  - libgomp=11.2.0=h1234567_1
-  - libiconv=1.16=h5eee18b_3
-  - libnghttp2=1.57.0=h2d74bed_0
-  - libpng=1.6.39=h5eee18b_0
-  - libssh2=1.11.1=h251f7ec_0
-  - libstdcxx-ng=11.2.0=h1234567_1
+  - libgcc=15.2.0=h69a1729_7
+  - libgcc-ng=15.2.0=h166f726_7
+  - libgomp=15.2.0=h4751f2c_7
+  - libmpdec=4.0.0=h5eee18b_0
+  - libstdcxx=15.2.0=h39759b7_7
+  - libstdcxx-ng=15.2.0=hc03a8fd_7
   - libuuid=1.41.5=h5eee18b_0
-  - libxcb=1.15=h7f8727e_0
-  - libxml2=2.9.14=h74e7548_0
-  - lz4-c=1.9.4=h6a678d5_1
-  - markupsafe=1.1.1=py27h7b6447c_0
-  - matplotlib=2.2.2=py27hb69df0a_2
-  - mkl=2020.2=256
-  - mkl-service=2.3.0=py27he904b0f_0
-  - mkl_fft=1.0.15=py27ha843d7b_0
-  - mkl_random=1.1.0=py27hd6b4f25_0
-  - ncurses=6.4=h6a678d5_0
-  - numpy=1.16.6=py27hbc911f0_0
-  - numpy-base=1.16.6=py27hde5b4d6_0
-  - openssl=3.0.15=h5eee18b_0
-  - pcre2=10.42=hebb0a14_1
-  - perl=5.38.2=0_h5eee18b_perl5
-  - pip=19.3.1=py27_0
-  - pyparsing=2.4.7=pyhd3eb1b0_0
-  - pyqt=5.6.0=py27h22d08a2_6
-  - python=2.7.18=h42bf7aa_3
-  - python-dateutil=2.8.2=pyhd3eb1b0_0
-  - pytz=2021.3=pyhd3eb1b0_0
-  - pyyaml=5.2=py27h7b6447c_0
-  - qt=5.6.3=h8bf5577_3
-  - readline=8.2=h5eee18b_0
-  - setuptools=44.0.0=py27_0
-  - simplejson=3.17.0=py27h7b6447c_0
-  - singledispatch=3.7.0=pyhd3eb1b0_1001
-  - sip=4.18.1=py27hf484d3e_2
-  - six=1.16.0=pyhd3eb1b0_1
-  - sqlite=3.45.3=h5eee18b_0
-  - subprocess32=3.5.4=py27h7b6447c_0
-  - tk=8.6.14=h39e8969_0
-  - tornado=5.1.1=py27h7b6447c_0
-  - wheel=0.37.1=pyhd3eb1b0_0
+  - libxcb=1.17.0=h9b100fa_0
+  - libzlib=1.3.1=hb25bd0a_0
+  - ncurses=6.5=h7934f7d_0
+  - openssl=3.0.18=hd6dcaed_0
+  - pip=25.2=pyhc872135_1
+  - pthread-stubs=0.3=h0ce48e5_1
+  - python=3.13.9=h7e8bc2b_100_cp313
+  - python_abi=3.13=1_cp313
+  - readline=8.3=hc2a1206_0
+  - setuptools=80.9.0=py313h06a4308_0
+  - sqlite=3.51.0=h2a70700_0
+  - tk=8.6.15=h54e0aa7_0
+  - tzdata=2025b=h04d1e81_0
+  - wheel=0.45.1=py313h06a4308_0
+  - xorg-libx11=1.8.12=h9b100fa_1
+  - xorg-libxau=1.0.12=h9b100fa_0
+  - xorg-libxdmcp=1.1.5=h9b100fa_0
+  - xorg-xorgproto=2024.1=h5eee18b_1
   - xz=5.6.4=h5eee18b_1
-  - yaml=0.1.7=had09818_2
-  - zlib=1.2.13=h5eee18b_1
-  - zstd=1.5.6=hc292b87_0
+  - zlib=1.3.1=hb25bd0a_0
   - pip:
-      - multiqc==1.0.dev0
-prefix: /home/schauerm/miniconda3/envs/MULTIQC
+      - annotated-types==0.7.0
+      - attrs==25.4.0
+      - boto3==1.40.69
+      - botocore==1.40.69
+      - certifi==2025.10.5
+      - charset-normalizer==3.4.4
+      - click==8.3.0
+      - coloredlogs==15.0.1
+      - colormath2==3.0.3
+      - humanfriendly==10.0
+      - humanize==4.14.0
+      - idna==3.11
+      - importlib-metadata==8.7.0
+      - jinja2==3.1.6
+      - jmespath==1.0.1
+      - jsonschema==4.25.1
+      - jsonschema-specifications==2025.9.1
+      - kaleido==0.2.1
+      - markdown==3.10
+      - markdown-it-py==4.0.0
+      - markupsafe==3.0.3
+      - mdurl==0.1.2
+      - multiqc==1.32
+      - narwhals==2.11.0
+      - natsort==8.4.0
+      - networkx==3.5
+      - numpy==2.3.4
+      - packaging==25.0
+      - pillow==12.0.0
+      - plotly==6.4.0
+      - polars-lts-cpu==1.33.1
+      - pyarrow==22.0.0
+      - pydantic==2.12.4
+      - pydantic-core==2.41.5
+      - pygments==2.19.2
+      - python-dateutil==2.9.0.post0
+      - python-dotenv==1.2.1
+      - pyyaml==6.0.3
+      - referencing==0.37.0
+      - regex==2025.11.3
+      - requests==2.32.5
+      - rich==14.2.0
+      - rich-click==1.9.4
+      - rpds-py==0.28.0
+      - s3transfer==0.14.0
+      - six==1.17.0
+      - spectra==0.1.0
+      - tiktoken==0.12.0
+      - tqdm==4.67.1
+      - typeguard==4.4.4
+      - typing-extensions==4.15.0
+      - typing-inspection==0.4.2
+      - urllib3==2.5.0
+      - zipp==3.23.0

env/env_nextflow.yml env/RNASEQ.ymlenv/env_nextflow.yml renamed to env/RNASEQ.yml

@@ -12,8 +12,9 @@ dependencies:
   - bioconductor-rsubread=2.0.0=r36h516909a_0
   - bwidget=1.9.16=h9eba36c_0
   - bzip2=1.0.8=h5eee18b_6
-  - ca-certificates=2024.7.2=h06a4308_0
+  - ca-certificates=2025.2.25=h06a4308_0
   - cairo=1.16.0=hb05425b_5
+  - coreutils=9.5=h5eee18b_0
   - curl=7.69.1=hbc83047_0
   - dbus=1.13.18=hb2f20db_0
   - expat=2.6.2=h6a678d5_0
@@ -27,7 +28,8 @@ dependencies:
   - gdbm=1.18=hd4cb3f1_4
   - gfortran_impl_linux-64=7.5.0=ha8c8e06_17
   - gfortran_linux-64=7.5.0=h96bb648_30
-  - glib=2.69.1=h4ff587b_1
+  - glib=2.78.4=h6a678d5_0
+  - glib-tools=2.78.4=h6a678d5_0
   - graphite2=1.3.14=h295c915_1
   - gsl=2.4=h14c3975_4
   - gxx_impl_linux-64=7.5.0=h0a5bf11_17
@@ -44,12 +46,14 @@ dependencies:
   - libcurl=7.69.1=h20c2e04_0
   - libdeflate=1.17=h5eee18b_1
   - libedit=3.1.20210714=h7f8727e_0
-  - libffi=3.3=he6710b0_2
+  - libffi=3.4.4=h6a678d5_1
   - libgcc-devel_linux-64=7.5.0=hbbeae57_17
   - libgcc-ng=11.2.0=h1234567_1
   - libgfortran-ng=7.5.0=ha8ba4b0_17
   - libgfortran4=7.5.0=ha8ba4b0_17
+  - libglib=2.78.4=hdc74915_0
   - libgomp=11.2.0=h1234567_1
+  - libiconv=1.16=h5eee18b_3
   - libpng=1.6.39=h5eee18b_0
   - libssh2=1.10.0=h37d81fd_2
   - libstdcxx-devel_linux-64=7.5.0=hf0c5c8d_17
@@ -61,20 +65,23 @@ dependencies:
   - libxml2=2.9.14=h74e7548_0
   - lz4-c=1.9.4=h6a678d5_1
   - make=4.2.1=h1bed415_1
-  - ncurses=6.2=he6710b0_1
-  - openjdk=11.0.13=h87a67e3_0
+  - ncurses=6.4=h6a678d5_0
+  - nextflow=24.10.5=h2a3209d_1
+  - openjdk=21.0.6=h38aa4c6_0
   - openssl=1.1.1w=h7f8727e_0
   - pango=1.50.7=h05da053_0
   - pcre=8.45=h295c915_0
+  - pcre2=10.42=hebb0a14_1
   - perl=5.34.0=h5eee18b_2
+  - picard=3.4.0=hdfd78af_0
   - pip=24.2=py310h06a4308_0
   - pixman=0.40.0=h7f8727e_1
-  - python=3.10.0=h12debd9_1
+  - python=3.10.11=h7a1cb2a_2
   - r-base=3.6.1=haffb61f_2
   - readline=8.1=h27cfd23_0
-  - samtools=1.10=h2e538c0_3
+  - samtools=1.6=h244ad75_5
   - setuptools=72.1.0=py310h06a4308_0
-  - sqlite=3.40.1=h5082296_0
+  - sqlite=3.45.3=h5eee18b_0
   - subread=2.0.1=h5bf99c6_1
   - sysroot_linux-64=2.17=h57e8cba_10
   - tk=8.6.14=h39e8969_0
@@ -84,4 +91,9 @@ dependencies:
   - xz=5.2.10=h5eee18b_1
   - zlib=1.2.13=h5eee18b_1
   - zstd=1.5.5=hc292b87_0
-prefix: /home/schauerm/miniconda3/envs/RNASEQ
+  - pip:
+      - cutadapt==4.9
+      - dnaio==1.2.2
+      - isal==1.7.1
+      - xopen==2.0.2
+      - zlib-ng==0.5.1

pipeline/conf/params.config

@@ -11,7 +11,6 @@ params {
     phageGFF                = "/input/reference/NC_000866-4.gff3"
 
     pairedEnd               = false //if calling --pairedEnd, read pairs will be collected
-    dRNAseq                 = false //if calling --dRNAseq, dRNA-seq-specific processing will be applied
 
     reads                   = "/input/reads/SRR21013140.fastq.gz"
     outputDir               = "/output"

pipeline/main.nf

@@ -4,7 +4,6 @@
 include { BUILDHISAT2BASE } from './modules/buildHisatBase'
 include { PROCESSRNASEQ } from './subworkflows/perReadProcessing'
 include { FEATURECOUNTS } from './modules/readCounting'
-include { PROCESSDRNASEQ } from './subworkflows/dRNAseqProcessing'
 include { CLEANUP } from './modules/cleanDir'
 include { MULTIQC } from './modules/multiQC'
 
@@ -28,7 +27,6 @@ def helpMessage() {
       --phageGFF              Phage genome annotation path in gff3 format.
 
       --pairedEnd             Default is false. Set, if paired-end reads are supplied.
-      --dRNAseq               Default is false. Would activate dRNA-seq specific processing. Currently not established!
 
       --adapter1              Default is sequence of TruSeq adapter.
       --adapter2              Default is sequence of TruSeq adapter.
@@ -49,13 +47,10 @@ workflow {
 
     BUILDHISAT2BASE(params.hostGenome, params.phageGenome, params.hostGFF, params.phageGFF)
 
-    if (params.dRNAseq) { //dRNA-seq specific processing
-        PROCESSDRNASEQ(params.reads, BUILDHISAT2BASE.out.alignmentBase)
-    } 
-    else { //RNA-seq processing
-        PROCESSRNASEQ(params.reads, BUILDHISAT2BASE.out.alignmentBase)
-        FEATURECOUNTS(PROCESSRNASEQ.out.sortedBamFile.collect(), BUILDHISAT2BASE.out.dualGFF)
-        MULTIQC(FEATURECOUNTS.out.countSummary, PROCESSRNASEQ.out.cutadaptReport.collect().ifEmpty([]), PROCESSRNASEQ.out.fastqc_pre.collect(), PROCESSRNASEQ.out.fastqc_post.collect())    
-    }
+    PROCESSRNASEQ(params.reads, BUILDHISAT2BASE.out.alignmentBase)
+    
+    FEATURECOUNTS(PROCESSRNASEQ.out.sortedBamFile.collect(), BUILDHISAT2BASE.out.dualGFF)
+    
+    MULTIQC(FEATURECOUNTS.out.countSummary, PROCESSRNASEQ.out.cutadaptReport.collect().ifEmpty([]), PROCESSRNASEQ.out.fastqc_pre.collect(), PROCESSRNASEQ.out.fastqc_post.collect())    
 
 }

pipeline/modules/multiQC/main.nf

@@ -16,6 +16,6 @@ process MULTIQC {
 
     script:
     """
-    multiqc . -n multiQC_report.html --no-data-dir -m custom_content -m preseq -m rseqc -m featureCounts -m star -m cutadapt -m fastqc -m qualimap -m salmon
+    multiqc . -n multiQC_report.html --no-data-dir -m custom_content -m preseq -m rseqc -m featurecounts -m star -m cutadapt -m fastqc -m qualimap -m salmon
     """
 }