Reverse reads - quality plot question

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#1

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

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#2

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
#3

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

#4

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

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#5

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

#6

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

#7

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