Hi @drmusk,
If you are following this protocol using V3-V4, the expected amplicon size is actually 460bp. Meaning on a 2x300 run you have 140bp overlap. DADA2 requires at least 20nt overlap for proper merging, so the maximum combined truncation to have effective merging should be ~ 120. You are truncating 80bp on both the forward and reverse reads, so 160bp. That means you do not have sufficient overlap and dada2 will fail to merge most if not all of your reads. Given the very poor quality of your forward reads, I would actually recommend just ditching those and using your reverse reads alone, try truncating those at 220 as you were. You will lose some resolution but you will retain most of your reads, besides I’m not sure if you have another option at the moment.
Let us know how that goes.
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