Deblur vs DADA2 Questions

[benjjneb (Ben Callahan)]

@antgonza

Removing adapters is something that I’m not even sure if someone has actually benchmarked in a programatic and extensive way. However, there are pros and cons about doing this. For example, on one side removing them will allow you to remove some “biases” during clustering, taxonomic assignments, etc (*) and not removing them will allow you to have a longer read, also biasing some of the other steps. Additionally, remember that normally your higher quality is at the beginning of the read.

The biggest problem arises from the ambiguous nucleotides in many primers. For example, the 515F primer is GTGYCAGCMGCCGCGGTAA. The two ambiguous nucleotides (Y=C or T, M=A or C) show up in equal proportions (technically there are a mixture of the 4 possible primers used).

As a result, if reads have the 515F primer on them, each real biological sequence will show up in a 25/25/25/25% mixture of the 4 possible primer sequences + real biological sequence. This is very bad when using ASV methods, as they will distinguish those differences and call 4 types for every 1 real type!

It's less of an issue if there are no ambiguous nucleotides, but still, primer/adapter isn't DNA from the sequenced organism. That why it is a plus for methods (e.g. EMP) that don't sequence the adapters/primers/etc, as they don't waste bases on non-bio DNA.