Dada2. Filtering steps discards most of my reads

Agreed! About 20% of the reads cannot be joined, but this is pretty good given the quality.

ITS classification is harder than 16S classification for a number of reasons. For a first run with a custom database (that you just made!), I think getting to order is pretty good!

Yes. It's all about classify-consensus-vsearch.

Aftering finding hits to the database with vsearch, the taxonomy of these hits are compaired. For each level where >50% of the hits agree a classification is given, until you reach a level where there is not longer a consensus.

For your data, the level in which hits no longer agree is mostly Order. :person_shrugging:


Your progress here is awesome. If I were you, I would keep these results and continue with the paper. If reviewer 3 thinks there is a better way to classify ITS-2, they will certainly tell you.

EDIT: If you do want to improve the database, try using RESCRIPt's 'extract-seq-segments', or adding a few "out-group" taxa to your database in case you amplified non-Magnoliopsida taxa.

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