Nextera adapters trouble – how short is too short a read

[Nicholas_Bokulich]

Hi @mpunta ,

@mpunta

Can I ask how you would measure taxa resolution? Would you simply look at percentage of assigned species, genus and all the way up the taxonomy for e.g. 150 vs 100 bp?

Exactly. The classifiers in q2-feature-classifier are meant to (by default) only classify to a depth at which the classifier is confident about the result (based on the reference data). This can be adjusted by changing the confidence threshold (classify-sklearn) or the consensus threshold (classify-consensus-* methods), e.g., to allow less confident classification (not necessarily a good thing!).

There is a QIIME 2 plugin, RESCRIPt, which can create a nice visualization of taxonomic resolution, e.g., number of unclassified taxa at each rank. See here for details:

Processing, filtering, and evaluating the SILVA database (and other reference sequence data) with RESCRIPt

Please consider this tutorial a living document, which may change based upon community feedback and ongoing plugin development. RESCRIPt RESCRIPt (REference Sequence annotation and CuRatIon Pipeline) is a python package and QIIME 2 plugin for formatting, managing, and manipulating sequence reference databases. This package was designed for compiling, manipulating, and evaluating sequence reference databases from SILVA, NCBI, Greengenes, GTDB, and other sources, and for construc…

@mpunta

And also what would you consider a satisfactory % at for example genus level?

The degree of resolution for any given taxon is going to depend on the length of the sequences and the marker-gene region. I suggest you just let yourself be guided by the results — unclassified sequences at phylum level are a sign of technical issues, but at genus level lack of classification is usually just lack of resolving power, about which there is little you can do (except choose a different marker gene or longer sequencing read length!), so there is not a satisfactory % for accepting/rejecting a result, only to accept/reject the technology! (and that threshold is decided by you )

I hope that helps!