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Old 03-23-2017, 09:17 PM   #21
nucacidhunter
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I have seen improvements in read qualities since early last year. Attached samples FastQC output for 16S amplicons with similar spike in and cluster density.
MiSeq 600 cycle kit quality improvement.pdf
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Old 03-23-2017, 10:03 PM   #22
fanli
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@nucacidhunter, was this an identical pool in terms of bacterial biomass and complexity? We've seen variation in sequencing quality for some amplicon libraries that probably had more off-target amplification due to low amount of true template.

It seems more cost effective to use the 600 cycle v3 kit in place of a 500 cycle v2 kit (and simply run a 2x250)...any thoughts?
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Old 03-23-2017, 10:14 PM   #23
nucacidhunter
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I have shown the general trend and these are amplicons prepared using two step PCR so the initial cycles for both read 1 and 2 includes primer sequences. Pool of 96 libraries are sequenced in one flow cell and because they are targeting the same region diversity always is low. I like V3 because the yields are higher than V2 reagents.
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Old 03-24-2017, 12:43 PM   #24
thermophile
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Wow that's a sig. improvement. We saw similar pattern last year with v3 as your first v3, so this is encouraging
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Old 10-24-2017, 04:33 PM   #25
jlli2000
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Does anyone have experience to run MiSeq v3 600 kit as r1 400 cycles and r2 200 cycles? The numbers may be variable.

Any feedback appreciated.

Thanks,

Jin




Quote:
Originally Posted by jhi_pete View Post
I have heard a rumour that the long standing MiSeq v3 600 bp kit chemistry problems which led to poor quality read 2 data have recently been solved by Illumina. Anyone know if this is true?
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Old 10-24-2017, 05:33 PM   #26
GenoMax
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@Jin: You should be able to run as you want without any issues (400 x 200 bp). Q-scores will likely tank on that 1st read later in the run.
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Old 01-16-2018, 06:15 AM   #27
ngagnes
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Hi everyone,

Illumina announced a Q30 performance back to the "normal" for the "long read" kits (600 cycles V3 and 500 cycles V2) since november 2017.
But since this announcement, I runned 4 runs 600 cycles V3 with still Q30 bad quality. Their tech support agreeded that it is still because of a sequencing kit reagents issue.

What about you? Are you run better since this date?
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Old 01-16-2018, 08:47 AM   #28
thermophile
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did tech support check your lot numbers to be sure that you were using the "new and improved" chemistry? Though I haven't been confident enough to try to convince a user to try the new v3 yet
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Old 01-16-2018, 04:55 PM   #29
luc
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Hi ngagnes,

in our experience the reagent quality has gotten better, but is still not as good as 3 years ago and we still see our low quality run outliers (that should have performed better according to cluster density). We are running almost exclusively amplicons.
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Old 01-17-2018, 04:27 AM   #30
ngagnes
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I sent them all the lot numbers, but they didn't give me the informations about "new or old chemistry". But the kit were sent on december, so I guess new one? This is why I was asking to have others feedbacks...
We are also running amplicons and small genomes too. And those 4 runs also had a good cluster density, so it is not because of over clustered runs.
Thanks for your replies, I hope that the Q30 quality will be better in few weeks!
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