recommendations for processing with poor quality forward reads

User Support
1

Hi, I am analyzing some 16S V4 rRNA amplicon data from low-diversity symbiont communities, planning on using DADA2 in QIIME2. The issue I am having is that the quality of the forward reads is very poor, while the quality of the reverse reads is also bad but more usable and typical for reverse reads I think. Example sequencing run:

image
image1718×536 181 KB

demux-paired-end.qzv (311.6 KB)

(the primer set results in an amplicon of ~400 bp, and the sequencing chemistry was 2x250 bp, so I'd expect ~100 bp overlap)

I'm not sure that DADA2 will be able to merge these, although I will attempt to run it in paired end mode and play with the parameters. But I suspect the forward reads might be too low quality to use at all, and I might want to denoise only the reverse reads. Is this possible in DADA2? I know that you can use only forward reads in the single end mode, but haven't found any info about using only reverse reads. I would appreciate any insight, thank you!

3

Hi @anna-schrecengost,

Thanks for reaching out and for providing those sequencing run screenshots! Here are a few thoughts on how to proceed here:

As of the QIIME 2 2021.4 release, DADA2 requires at least 12nt overlap for proper merging - so that's something to keep in mind if you only have ~100 bp of overlap.

I do agree with you that your forward reads are pretty low in quality, and may not be good enough to use at all (it appears as though the only semi-decent quality score is from 0-10 in your sequence base, which is pretty short).

Using only reverse reads in denoise-single isn't typically recommended due to the commonly lower quality scores of reverse reads, but in this case it does seem like your best bet. In this forum post, this is what @Mehrbod_Estaki recommended to another user who has poor quality forward reads as well. Hopefully this helps!

Cheers,
Liz

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Thank you @lizgehret!