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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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