Separating two different amplicons from demultiplexed data

[Nicholas_Bokulich]

@JeanRodrig

Would this approach still work if my sequences were dual indexed for sequencing and were demultiplexed by the sequencing facility?

Sorry, missed that point yesterday — if the data are already demultiplexed, you cannot go the first route. Would need to run dada2 and then use exclude-seqs later. Or get the raw, multiplexed data from the sequencing center if that's even possible.

@JeanRodrig

Primers are contained in the sequences but each sample’s R1 and R2 file contain sequencesbelonging to the two different reads thus, two different sets of primers. I’m a newbie in the topic and might be missing the point but seems like all the options in q2-cutadapt are for single barcodes.

q2-cutadapt can still be used to trim the primers from each end on dual-indexed reads with the trim-paired method. You are correct, though — demux-paired cannot handle dual-indexed reads for demultiplexing.

@JeanRodrig

If q2-cutadap is not an option, then exclude-seqs might be the way to go after dada2, using the alignment as the excluding factor

@JeanRodrig

can I run dada2 with the sequenced containing primers?

You can, but you should not if you are using degenerate primers. Instead:

1. Use q2-cutadapt trim-paired to trim primers twice (once for each of your two primer sets)
2. Run dada2
3. Use exclude-seqs to exclude by alignment against each reference database. No primers required.