Cutadapt and sequence classification.

[Nicholas_Bokulich]

Welcome @rsak-384!

@rsak-384

• Can the ‘qiime cutadapt trim-paired’ plugin handle degenerate primer bases. For example: can i provide the whole 341F primer (CCTACGGGNGGCWGCAG) for the ‘–p-adapter-f’ parameter or should i only provide the part with no degenerate bases (CCTACGGG)?

Sure — here are some relevant parameters for you:

  --p-error-rate PROPORTION Range(0, 1, inclusive_end=True)
                          Maximum allowed error rate.           [default: 0.1]
  --p-match-read-wildcards / --p-no-match-read-wildcards
                          Interpret IUPAC wildcards (e.g., N) in reads.
                                                              [default: False]
  --p-match-adapter-wildcards / --p-no-match-adapter-wildcards
                          Interpret IUPAC wildcards (e.g., N) in adapters.
                                                               [default: True]

@rsak-384

Kind of a similar question the the first one. Can the ‘qiime feature-classifier extract-reads’ plugin handle primers with degenerate bases in them or should i provide only the part of the primer with no degenerate bases?

Yes, see here:

Allowed Primer-read mismatch in extract-reads?

HI @jwdebelius, @Mehrbod_Estaki, extract-reads handles degenerates correctly. The local aligner from skbio that we use has some … interesting behaviour regarding degenerates, but from memory we worked hard to work around them. The answer to your question is in —p-identity: --p-identity NUMBER minimum combined primer match identity threshold. [default: 0.8] So the mismatch threshold is defined as a fraction of the combined …

@rsak-384

1. Should one cluster the feature table to ‘–p-perc-identity 97/99’ using ‘qiime vsearch cluster-features-de-novo’ before using the ‘qiime feature-classifier classify-sklearn’ plugin? What could be the advantages/disadvanges of doing so? Feature table prepared with ‘DADA2’.

If you've used dada2 to denoise, there is no need to cluster into OTUs. I recommend checking out the "overview" tutorials at tutorials.qiime2.org for some notes on advantages and disadvantages, and even better is probably the community discussions... see some OTU + denoising topics here:
Frequent Questions and "Best of the QIIME 2 Forum"

I hope that helps!