Reverse reads - quality plot question

Hi Everyone,

I'm analyzing a new dataset, and, after importing the data into Qiime2 I saw that the quality plot of my reverse reads drops to almost zero and, afterwards, increases.

I'm just wondering why this happened. When I denoise the data (using DADA2), should I trim the reverse reads necessarily up to 180 bp?

Thanks very much,
FS

Hello Fernando,

Yes, trimming at 180 would probably be your best option. Your second best option would be to contact your sequencing center, report this as a failed run, and ask them to run it again. It’s possible (hopefully!) that this is some sort of software error and your run is OK, but if I saw that graph, I would see if they could rerun those samples. :disappointed_relieved:

Let’s see what the dada2 folks recommend.

Colin

3 Likes

I agree with @colinbrislawn, sorry about your data @fstudart :frowning:

Hi Dr. Colin,

Thanks for your feedback. I'm also including the forward plot for your analysis, and I did not see the same artifact with these reads. So, may I assume that this sequencing problem was limited to the reverse reads? Also, when I merge the reads and considering that to merge is only necessary a 20-bp overlap, by trimming the reverse reads up to 180 bp, would it be ok to proceed?

Thanks,
Fernando Studart

Hi Dr. Bolyen,

Thanks for your reply. I just included the forward plot as well. Do you think I can proceed analyzing this run, as I did not see the same artifact with the forward reads?

Thanks,
FS

Hello again,

Yes, that sounds right. And it’s common for Illumina data to have issues with one direction.

Yes! This is why paired end reads are soon good! I think your analysis will not even be effected by this region.

Good luck!
Colin

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