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Again, this is only for the Illumina 16S V3 and V4 region amplicons. If you’ve amplified a different region, you’ll need to provide different primers. If you’re using Illumina, look out for overhang sequences!
Running ampliseq on pacbio data
ampliseq is designed for paired-end Illumina data, but can be run on single-end pacbio data with a few modifications:
Manifest file
A paired-end manifest requires exactly ‘sampleID forwardReads reverseReads’ as column names.
For single end just use ‘sampleID Reads'.
See line 349 of the main.nf file for single_end samples: .map { row -> [ row.sampleID, file(row.Reads, checkIfExists: true) ] } compared to the default paired-end line: .map { row -> [ row.sampleID, [ file(row.forwardReads, checkIfExists: true), file(row.reverseReads, checkIfExists: true) ] ] }
https://github.com/nf-core/ampliseq/blob/master/main.nf
nextflow.config
Add a line ‘pacbio = true’
This tells ampliseq to run using single_end parameters and also changes some of the DADA2 parameters.
Running NextFlow’s ampliseq pipeline
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