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  • #16
    We did a 2x300 run for a TCR library recently. Passed spec (77.5% Q30 @ 939k with 15% PhiX), but clear tailing towards the ends of both R1 and R2 as well.

    I could see error rates approaching 5% being problematic for many if not most applications.

    We'll be sticking with 2x250 or 2x150 chemistry for the time being, unless specifically requested by a client.
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    • #17
      To my knowledge error rate is not included in Illumina specifications. Q scores drop after cycle 250 is a known characteristic of the chemistry and for some applications it will not be detrimental.

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      • #18
        So.. no this issue is not resolved. I have an email from Illumina today which I will summarise.

        The issue is that there is a byproduct in their manufacturing process of fluorescent nucleotides. This byproduct results in NON-CLEAVABLE NTPs - so they limit strand growth. Obviously this has limited effects on the number of clusters per cycle, but does become more acute on 600 cycle kits as the problem 'builds'. You get a lower cluster density, and lower Q30 and higher error rates on the later cycles.

        Illumina can now *detect* this byproduct and know what levels impact performance, which sounds to me like there might be batch to batch variation, and have put in place measures to screen for it, therefore they are still expecting improvements 'in the near future' as newer material hits the manufacture pipeline. I am also told there will be a new announcement soon, with new timelines for resolution.

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        • #19
          thanks for the update

          I've have also heard that the problem wasn't resolved, but never had details as to the cause.

          Thanks!

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          • #20
            Many thanks Bukowski, that's the first logical explanation I have heard. Looking forward to any announcements from Illumina going forward.

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            • #21
              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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              • #22
                @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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                • #23
                  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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                  • #24
                    Wow that's a sig. improvement. We saw similar pattern last year with v3 as your first v3, so this is encouraging
                    Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

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                    • #25
                      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




                      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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                      • #26
                        @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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                        • #27
                          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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                          • #28
                            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
                            Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

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                            • #29
                              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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                              • #30
                                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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