I have demultiplexed paired-end reads that were amplified with the igk3/dvv primer pair, which has been used to taxonomically classify diazotrophic communities. These primers:
IGK = GCNWTHTAYGGNAARGGNGGNATHGGNAA
DVV = ATNGCRAANCCNCCRCANACNACRTC
Could be argued to be fairly degenerate, especially considering the number of N's in either primer
When I used the dada2 denoise-paired command, however, of the 90,000 forward and 90,000 reverse reads I originally had, only 3 sequences pass the filtering steps, as it appears that an overwhelming majority of my sequences are considered chimeras, although I think that might be because of the primers I used were degenerate, so at least some of my sequences might be confused as chimeras.
I've tried several ways to work around this issue, such as trying to retain borderline chimeras (Identifying and filtering chimeric feature sequences with q2-vsearch — QIIME 2 2021.4.0 documentation), although I think that requires that my chimeras be retained after DADA2. So, I've tried using commands such as --p-chimera-method none (just as it's written, changing it from the default consensus) or --p-min-fold-parent-over-abundance 8 (just as it's written, changing it from 1), but the chimera filter apparently remains on, as only 3 sequences still pass the filtering.
I've attached the original results from DADA2 (without tweaking with the chimera detection methods). Any advice anyone has on how to deal with this issue would be greatly appreciated. Thank you!!