MiSeq: Sudden drop in %> Q30 halfway read 1

[vehuardo]

We have a run going on the MiSeq as we speak (2x250 bp). I just had a look at the run in the SAV.

A sudden drop in Q30 after about 75 cycles caught my attention. Has anyone seen anything like this?

*Pic attached*

[bryanbriney]

How are you preparing your library? We do a lot of amplicon sequencing, and sharp drops in quality in the 75-150bp region typically indicate some level of PCR artifact (primer-dimer) in the library. Since the artifacts have adapters, they load and sequence, but once the short fragment is done being sequenced (about 75-150 cycles into the run) the quality score for those clusters drops sharply.

Short fragments tend to load preferentially, so even a relatively small amount of artifact can have a pretty large effect on the run.

[vehuardo]

Thanks Bryanbriney.

These were whole-genome frgmented DNA libraries prepared using Kapa + bioo scientific indexes. You are probabbly spot on. There were some adapter-dimers and adapters visible on the bioanalyzer trace. Priot to this we only used gels, so we were unsure whether these adapters were something we had had also in previous runs but failed to spot on the gels. In that case, as previous runs have gone well, we decided to sequence and see what happens. Probably less than perfect AMPure clean-up after adapter ligation and PCR then....

I guess doing another clean-up of the samples pooled after PCR could be a good option, and then start another run...

[aerseq]

Check out this thread:

http://seqanswers.com/forums/showthread.php?t=59558

Illumina has acknowledged an issue with their MiSeq 600 cycle v3 kits, though it has not been resolved yet.