DADA2 filtering out most of my reads


I am working on 16S data (primer 515FB/806RB, paired end) that is 92 libraries in one MiSeq run that has been demultiplexed and adapters removed in qiime2. At the DADA2 step, around 40 of my libraries are being filtered out in the initial filtering step ("The filter removed all reads"). I have looked at the sequence length distribution for all our libraries and many of our forward reads are pretty short so I am assuming they are being removed because there is not enough overlap with the reverse read, but some of the libraries being thrown out should be the correct length. I was hoping you could provide some insight as to how this filtering step works.

I have attached my .qzv file from after the trimming stage. As you can see we had some issues with uneven sequencing across our libraries and would like to resequence but we would like to know as much as possible about our current data and the qiime2 pipeline before sequencing again.

Thank you!

trimmed-seqs-anywhere-noA3-112-ALL.qzv (295.2 KB)

Thanks for the summary viz, that was helpful, @deefach! The first thing that jumped out at me there is that according to that summary, your forward reads contain zero-length sequences (yikes!) - that is a bummer.

I can’t really provide any specifics here until we get a sense of the parameters you provided to DADA2 during this step - can you please copy-and-paste the exact command or commands you ran? Otherwise, have you had a chance to look at the DADA2 documentation? DADA2 is an independently developed tool - it is exposed in QIIME 2 via the q2-dada2 plugin. Take a look at the documentation I linked to, and if you still have questions please provide us with the commands you ran. Thanks! :t_rex:

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