...
Then run the nfcore/ampliseq test workflow, which runs on a small test dataset.
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module load java
nextflow run nf-core/ampliseq -r 2.9.0 -profile test,singularity --outdir results
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...
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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:
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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" -- |
...
FW_primer "GGATTAGATACCCBRGTAGTC" --RV_primer "TCACGRCACGAGCTGACGAC" --outdir results
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...
This 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" The samplesheet 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.
The workflow takes approximately 40 minutes to complete.
Running again, with metadata
In the previous run to save time we didn’t include the dummy metadata. You can run it in the background now with the metadata option included, to see how it runs.
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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_with_metadata
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Additional parameters:
--metadata "data/metadata.tsv" Include the metadata file
--outdir results_with_metadata Output results to a new directory, called results_with_metadata
Interpreting ampliseq results
https://nf-co.re/ampliseq/2.9.0/docs/output
Z:\meta_workshop\illumina\results