V3-V4 amplicon trimming adapter

[Nicholas_Bokulich]

Welcome back, @arlandan,

@arlandan

• Primer sequences are retained in the V3-V4 amplicons. Is it necessary to trim the primer sequence off? I am assuming not, since they are at 5’ end, right?

For dada2 it is always necessary to trim the primers, see here for more info.

@arlandan

1. the stats file generated from dada2 denoising step have percentage of input filter/denoised/merged/non-chimeric values. how do you evaluate these numbers? The higher the better. what is considered as good enough? any minimums? Thank you very much.

It is all fairly subjective, based on your expectations and requirements. In general, filtering at the "filter" step is okay as long as you are getting a reasonable number of reads out the other end, but losing reads at the "merge" step is bad news, as this will bias the taxonomic composition of your samples (since shorter amplicons will be selectively excluded). See this post for a little "guide" I put together as a rule of thumb for interpreting read loss with dada2:

conceptual justification of dada2 truncation prior to merging

I can’t be sure without seeing some data and getting some specifics on what you did, but it sounds like the issue here is probably related to trimming low-quality bases from the 3’ ends of the reads, causing the reads to fail merging. Not because dada2 is dropping all reads due to the presence of errors (but it could be that too, I need to see the specifics). If you want to get our help troubleshooting that issue, please open a new topic and provide the usual info about what you ran, the qualit…

I hope that helps!