Hi @LiyingXie, I would like to add that since your primers contain IUPAC ambiguity bases, e.g. H, V, W. I would highly recommend that you add the following flags: --p-match-adapter-wildcards, and --p-discard-untrimmed. That is if the former flag is not enabled, only ATGC matches will be allowed, any ambiguous bases will count as a mismatch resulting in cutadapt in not “finding” the primer sequences, thus not removing it.
The latter flag, will discard any sequences in which you can not find the primer. I like to use this as a form of quality control, as it’ll remove sequences in which the primer could not be found. Otherwise, the sequence will remain in your data and can affect your downstream analysis.
You likely do not need --p-match-read-wildcards, but it wouldn’t hurt to use that in too.
-Cheers!
-Mike