Duplicates causing issues, how to handle merging and clustering?

[colinbrislawn (Colin J Brislawn)]

Hello Ravio,

Welcome to the forums!

This is a great question! We just spoke about ASVs vs OTUs for ITS amplicons in this thread:

ASV vs OTU for fungal ITS

Hello Everyone, I am searching for some more clarity about the use of ASV or OTU for fungal ITS analysis. After going through many publications I am seeing comments against the use of ASV for fungal ITS where the main rationale is that 1) ITS is variable in length and not suitable and 2) Fungal ITS show intraspecific and intragenomic differences and thus ASV ITS sequences must be clustered to approach species level resolution. While in my opinion the use of ASV seems like a good option cons…

This context may be helpful, as the consensus in this community is to do as much as possible at the ASV level. You will have to decide what setup works best for your data.

@raivo-otus

As I'm working with ITS sequences, I have understood they benefit greatly from a clustering step before taxonomic classification

Good news: taxonomy classification works equally well on any sequences (OTUs or ASVs). The benefit of converting your ASVs into something else is extremely dubious from my perspective.

@raivo-otus

dada2 denoising will be run on the combined sequences across 11 sequencing runs and not on each sequencing run individually, potentially affecting error rate learning.

Correct. DADA2 was designed to be run once on each sequencing run.