MiSeq: R1 vs. R2 reads show big quality difference - is that normal?

Question

[mschmid]

Hi!

A company sequenced 20 different samples for our lab on MiSeq platform (2x300). Now I got the data and did a first rough evaluation of the quality. I have huge differences in quality of R1 reads vs. R2. Is that normal (see images)?

The two images show first the R1 reads an then the R2 reads. I averaged the prob. for a wrong basecall over all reads of one sample.

On the y-axis you can see the probabilty of a wrong base-call and on the x-axis the base pos. Be aware of the fact that the two y-axes do not have the same range (otherwise one would not see anything in R1 graph).

Important: At graph for R2 the x-axis is flipped (Did this already in a script to align them later...)

http://picpaste.com/pics/R1.1398695966.png

http://picpaste.com/pics/R2.1398696015.png

Thanks!

Michael

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OK, here the phred scores. This time the x-axis of R2 is not flipped. What do you guys think? Is this a satisfying result considering that sequencing is done by professionals?

http://picpaste.com/pics/R1_phred.1398701258.png

http://picpaste.com/pics/R2_phred.1398700926.png

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Two additional infos:

First, the distribution of phred scores (Image 1: R1, Image 2: R2). Here I have just counted the occurrence of phred scores per sample:

http://picpaste.com/pics/R1_Phreds_1.1398765452.png

http://picpaste.com/pics/R2_Phreds_1.1398765471.png

Second, I counted how many reads pass the following criteria:

150bp with at least a average phred score of 20 in a moving window of 3 bases (I guess that is not very strict criteria).

For R1 90% to 95% of the reads pass

For R2 just about 0.01% pass

[DG]

I usually do see differences between read 2 and read 1 of a pair in terms of quality, but usually not that bad.

[brentp]

It would be interesting to see these charts only for bases with a quality > 20 or 30. Presumably, you axis is flipped on the R2.

[mschmid]

@ brentp: Yes sure! Stupid me, forgot to mention that... But still the R2 reads are much worse compared to R1 reads on the whole lenght... Guess that is still strange?

[lelle]

As Dan Gaston already said it is normal to see some difference, but I assume also that this is more than what one would expect. It is hard to say because I normally look at the Phred score directly without computing error probabilities. When I have seen a big difference between read 1 and read 2 quality this normally also means that the second index read is really low quality with a lot of uncalled bases. Did you have index reads and how do they look?

[JC]

I second previous comments, it's normal to have a small variation, R2 is in general a little bad compared to R1, but your plots shows that something happen in R2 sequencing. Ask for a re-run or ignore R2 in your analysis.

[brentp]

It doesn't seem that odd to me that at the end of 300 bases in the 2nd pair in untrimmed reads, the error rate is up to 10%. But I usually don't look at the data like this so maybe I'm mistaken.

[DG]

I agree. I usually look at it plotted out in the same way as FastQC does.

Answer 1

[umer.zeeshan.ijaz]

Hi mschmid,

Yes, it is normal for MISEQ to have R2 reads worse in quality than R1 reads. I play with MISEQ a lot and observe the same trend quite often. For reference, I recommend you to visit my page where I have compared different runs for average quality between R1 and R2 for MISEQ:

http://userweb.eng.gla.ac.uk/umer.ijaz/bioinformatics/oneliners.html?#SPATIAL

Also check Page 10 of these slides (my supervisor presented them at STAMPS 2013):

https://stamps.mbl.edu/images/9/9a/NoiseRemoval2013.pdf

Best Wishes,
Umer

[brentp]

cheers, those are very helpful analyses that you linked in your "one liners" page.