Quality Drop In The Reverse Read Of Paired End Library (Illumina Hiseq 1000)

Question

[ancient_learner]

Hi Recently we have prepared 3 paired end libraries (2X100) and sequenced on illumina hiseq 1000. we have checked the quality of the sequenced reads and found that for one library both forward and reverse reads showed good quality fatsQC reports. However for other 2 libraries the fastQC report for forward read (R1) is very good. But the fastQC report for reverse read is looking really bad (especially the per base quality score). I suppose if there is any problem with the library preparation then it should affect both the reads (R1, R2) which is not the case here.Could anyone let me know the reason why this is happening? I have posted this in seqanswers as well.

Thanks

[JC]

That sounds to me like a problem in the machine or the second strand synthesis, you can ask to your sequencing provider for a re-run or a refund.

Answer 1

[Ido Tamir]

The read quality has very little to do with library preparation. The sequence content influences the basecall qualities e.g. 100%G on one or even more positions or homopolymers lead to a drop in qualities. If your reads don't overlap, then you would have different qualities in read1 and read2. If your 3 samples are similar and don't have these problems as the good quality of one of your libraries indicates, its a problem of the run chemistry/readout.