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dos2unix $HOME/meta_workshop/illumina/data/metadata.tsv

Running nfcore/ampliseq

Run the full nfcore/ampliseq by copying the following into PuTTy:

cd $HOME/meta_workshop/illumina
module load java
nextflow run nf-core/ampliseq -r 2.9.0 -profile singularity --single_end --ignore_failed_trimming --input "data/samplesheet.tsv" --metadata "data/metadata.tsv" --FW_primer "GGATTAGATACCCBRGTAGTC" --RV_primer "TCACGRCACGAGCTGACGAC" --outdir results

ParametersThis moves to your $HOME/meta_workshop/illumina directory, loads the java module (Nextflow needs this) and runs the full ampliseq workflow, with all the parameters.

The parameters:

-r 2.9.0 runs version 2.9.0 of the ampliseq workflow. This is important for version control.

-profile singularity is the type of container we use on the HPC. Nextflow uses containers to run.

--single_end Since we have single-end data, we need to add this parameter. If we had paired-end we don’t need to add anything as paired-end is the default.

--ignore_failed_trimming Some of the samples in the public dataset are poor quality and fail the adapter trimming step. We’re ignoring these in this practice session, but if you have your own dataset you’ll want to address this in other ways (e.g. re-sequence samples, remove as outliers, etc).

--input "data/samplesheet.tsv" --metadata "data/metadata.tsv" The samplesheet and metadata files you created. Note in this case they must be in a ‘data’ subdirectory, but they can be anywhere you like, which you should then provide the full path for.

--FW_primer "GGATTAGATACCCBRGTAGTC" --RV_primer "TCACGRCACGAGCTGACGAC" The forward and reverse primers used. This is from the paper.

https://www.mdpi.com/2073-4425/11/9/1105

The hypervariable V5 and V6 regions (276 base pairs—bp) of the 16S rRNA gene were amplified using the 785F (5′-GGA TTA GAT ACC CBR GTA GTC-3′) and 1061R (5′-TCA CGR CAC GAG CTG ACG AC-3′) primers [20]

--outdir results The output directory for results. You can call this whatever you like.