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Old 12-06-2019, 05:15 AM   #110
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Location: Atlanta

Join Date: Feb 2014
Posts: 1

Originally Posted by dsobral View Post
Just to add our 2 cents.

We have a MiSeq running since 2013, and after some hickups we're now stable with it and reasonably happy.

We just recently installed a NextSeq500 and our first tests are not great. Q30 is >80%, but there are many low quality bases (constantly Q=14 "/"), and the worst part is that instead of being towards the end, they seem a bit randomly distributed. When comparing PhiX in a 2x150bp NextSeq with a 2x250bp MiSeq, after alignment I see a 0.2-0.3% error rate with MiSeq and 0.9-1% error rate with NextSeq (1M sampled reads). In the "randomly" distributed Q=14 bases I seem to notice more A to T transitions, but I didn't have time to gather more systematic statistics... If I do quality trim on the MiSeq I can easily get higher quality data, with the NextSeq since its randomly distributed is harder...

We've complained to the Illumina people, let's see what they say...
Were you/anyone else ever able to resolve this issue with random Q14 scores throughout the length of the read? We currently see the same in our data.
Our core recently mothballed our HiSeq2500 due to outrageously high service contract costs. We have converted to a NextSeq500 running NextSeq control software v2.2.0.4, RTA v2.4.11. We are running Blue Pippin selected multiplexed Nextera XT libraries (we are an environmental microbiology lab so we can have very low nucleic acid yields so we stick with XT) for 300 cycle PE High output runs . I'm told we can get around this random low quality issue for metagenome assembly as long as the quality scores of the other bases are true and we sequence deep enough but we are also interested in SNP analysis on some of our other projects and this would be problematic (I don't do these analyses myself-I'm on the front end of library making and sequencing). Our MiSeq is also a bit aged and misbehaving so currently on-site we only have access to instruments running two-dye chemistry. One problem that we seem to see consistently on our NextSeq besides the data quality (now for 3 runs) is that the green channel intensity is consistently low. Illumina tells me that this is not good and could be the camera. My core tells me that this issue only occurs on my runs (they seem to blame the Nextera XT). Any help/insight would be appreciated.
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