@ebolyen, would you say qiime cut-adapt demux-paired should still be applied after qiime demux emp-paired just to remove adapters
does demux emp-paired actually removes barcode while demultiplexing which are generally placed after the adapters?
Thank you for the good work
No, you wouldn’t demultiplex twice, you might use cutadapt trim-pairedif 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.