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Overview of steps in AMR++ with merged reads

Confirm everything works

You'll need to load the typical AMR++ environment, plus add "FLASH" and "SeqKit". Or you can simply re-install the AMR++ conda environment using the recipe as shown in the installation tutorial.

You'll still need to run the "demo" as this downloads the github with the SNP confirmation software. If you forget to do this, make sure your sbatch script includes the module that allows internet connection (e.g. "module load WebProxy").

Download a new AMR++ repository and switch to "dev" branch

git clone https://github.com/Microbial-Ecology-Group/AMRplusplus

cd AMRplusplus/

git pull origin dev

Full pipeline

You can run the full AMR++ pipeline (without kraken) by specifying the path to your --reads and using the --pipeline se_AMR flag. This could work well if you re-direct the results or the work directory -w /path/to/shared_drive.

Step 1 : Run the "eval_qc" pipeline as normal with --reads

Example command:

nextflow run main_AMR++.nf --pipeline eval_qc --output SE_AMR++_analysis --reads "data/raw/*.fastq.gz" -profile local

Step 2: Run "se_trim_qc" pipeline with --reads

Modify trimming parameters as needed in the params.txt file. Notice the --reads flag does not require a regular expression pattern to match paired reads, we can just use the asterisk "*" and file extension name to point to all samples.

Example command:

nextflow run main_AMR++.nf --pipeline se_trim_qc --output SE_AMR++_analysis --reads "data/raw/*.fastq.gz" -profile local

Step 3: Run "se_rm_host" with --reads

Parameters to change

Parameters that have to change:

  • --pipeline ==> --pipeline rm_host
  • --reads ==> --reads "SE_AMR++_analysis/HostRemoval/NonHostFastq/*.fastq.gz"
  • host ==> --host "/path/to/your/host/chr21.fasta.gz"
    • remember, you can change this in params.config file or add it to your nextflow command.
    • On grace, bovine: /scratch/group/big_scratch/SHARED_resources/host_genome/GCF_002263795.3_ARS-UCD2.0_genomic.fna

Example command:

nextflow run main_AMR++.nf --pipeline se_rm_host --output SE_AMR++_analysis --reads "SE_AMR++_analysis/HostRemoval/NonHostFastq/*.fastq.gz" -profile local

Step 4: Run "se_resistome" and point to non host reads with --reads

Parameters that have to change:

  • --pipeline ==> --pipeline se_resistome
  • --reads ==> --reads "SE_AMR++_analysis/HostRemoval/NonHostFastq/*.fastq.gz"

SNP confirmation and alignment deduplication is performed by default.

Example command:

nextflow run main_AMR++.nf --pipeline se_resistome --output SE_AMR++_analysis --reads "SE_AMR++_analysis/HostRemoval/NonHostFastq/*.fastq.gz" -profile local

Optional - Step 5: Run "se_kraken" and point to non host reads with --reads

Parameters that have to change:

  • --pipeline ==> --pipeline se_kraken
  • --kraken_db ==> --kraken_db /path/to/your/kraken_db

Parameter still pointing to nonhost merged reads

  • --reads ==> --reads 'SE_AMR++_analysis/HostRemoval/NonHostFastq/*.{merged,unmerged}.non.host.fastq.gz'

Example command:

nextflow run main_AMR++.nf --pipeline se_kraken --output SE_AMR++_analysis --reads "SE_AMR++_analysis/HostRemoval/NonHostFastq/*.fastq.gz" --kraken_db "/path/to/your/kraken_db" -profile local