How to remove primers and contaminants when using two pairs in multiplex?

[SoilRotifer (Mike Robeson)]

Hi @vln21, welcome to !

You can simply enter multiple primers as summarized here:

q2-cutadapt output

Good news @jessica.song! I think I solved this. First, thank you for sending me the PDF describing the protocol. I noticed a key statement: pairs of primers (Fw-Rev or Rev-Fw) had to be present in the sequence fragments That is, this particular sequencing facility must expect mixed orientation reads. Therefore you need to enter in both primers for each of the --p-front-* commands (in 5' - 3' orientation). Like so: qiime cutadapt trim-paired \ --i-demu…

More specifically, you can try the following:

qiime cutadapt trim-paired \
  --i-demultiplexed-sequences paired-end-demux.qza \
  --p-cores 4 \
  --p-front-f GATCGAGAAGACCCTRTGGAGCT  GACGAGAAGACCCTAWTGAGCT \
  --p-front-r AAATTACGCTGTTATCCCT  GGATTGCGCTGTTATCCCT \
  --p-match-read-wildcards \
  --p-match-adapter-wildcards \
  --p-discard-untrimmed \
  --o-trimmed-sequences paired-end-demux-primer-trimmed.qza \
  --verbose > cutadapt-log-2.txt

Then you can denoise.