Clarify on demux emp-paired

[WAS1]

Continuing the discussion from Demultiplexing paired end read, trimming adapters and barcodes:

@ebolyen

You’ll want to use qiime demux emp-paired .

Which primers were used doesn’t matter to this step, what’s more important is that your data has the non-biological data already stripped. In other words, the linker, primer, barcode, etc, are not in R1 or R2 (barcodes should be in R3 ). It will use the order of R3 to work out which sequences from R1 and R2 belongs to which samples.

qiime cutadapt demux-paired is for when your barcodes are still in the sequences and need to both be identified and trimmed out. This shouldn’t be the case with an EMP-like protocol.

@ebolyen, would you say qiime cut-adapt demux-paired should still be applied after qiime demux emp-paired just to remove adapters
or
does demux emp-paired actually removes barcode while demultiplexing which are generally placed after the adapters?
Thank you for the good work

[ebolyen (Evan Bolyen)]

Hey @WAS1!

@WAS1

@ebolyen, would you say qiime cut-adapt demux-paired should still be applied after qiime demux emp-paired just to remove adapters

No, you wouldn't demultiplex twice, you might use cutadapt trim-paired if your sequencing protocol was not EMP (but sufficiently similar that emp-paired successfully demuxed your reads). I don't think I've seen that before.

Otherwise, using cutadapt demux-paired will remove the barcodes, but not necessarily the primer.

Setting a trim-left parameter in dada2 is usually sufficient since the primer is of a known length. In some cases you might need to get rid of your reverse-complemented reverse-primer on your forward reads (and vice-versa) if your amplicon is sufficiently variable that you tend to sequence over the reverse primer in the forward direction. That's where cutadapt trim-paired is handy.