Removing primers with degenerate codes

Question

[life99945]

Hello!

I have forward and reverse illumina myseq 16s rRNA v4-v6 amplicon reads with Pro-mod-341F 5’-CCTAYGGGDBGCWSCAG and Pro-mod-805R 5’-GACTACNVGGGTMTCTAATCC primers used. I want to make sure these primers are already removed. Is there any way to do it?

Thank you.

Answer 1

[h.mon]

Use bbduk.sh from the BBMap / BBTools packages to search for the primers:

bbduk.sh in=B1_sub_R1.fq.gz literal=CCTAYGGGDBGCWSCAG copyundefined=t mm=f \
  ktrim=l k=13 mink=3

You can also use bbduk to trim the primers, see an example at this dada2 tutorial (but note I think you need copyundefined=t):

bbduk.sh in=B1_sub_R1.fq in2=B1_sub_R2.fq \
  out=B1_sub_R1_trimmed.fq.gz out2=B1_sub_R2_trimmed.fq.gz \
  literal=GTGCCAGCMGCCGCGGTAA,GGACTACHVGGGTWTCTAAT k=10 ordered=t mink=2 \
  ktrim=l rcomp=f minlength=220 maxlength=280 tbo tpe

[life99945]

Thank you very much!

[life99945]

Thank you very much, everything worked out, but I do not understand why in the example they don’t use copyundefined? After all, it greatly affects the reverse read trimming. And how can i count minlength? In example they say that "min and max length were based roughly on 10% smaller and bigger than would be expected after trimming the primers", but how can i expect to get 220 bp after trimming 17bp from 300bp?