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  • #16
    That is enough votes now.

    Like Phillip/Scott, we had assumed that this was a "feature" of the low diversity libraries we get a lot. There were no complaints so there were no immediate red flags. I would say this is going on for at least 6+ months. I am going to have to go back and dig though past flowcells tomorrow.

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    • #17
      Originally posted by pmiguel View Post
      Actually that is pretty much what all 600 cycle runs seem to do. Oddly 500-550bp amplicons seems to PANDA merge just fine, so I tended to assume that the instrument was just losing its confidence. But since the phiX error rate also tends to sky rocket at this point, I am not sure what to think.

      --
      Phillip
      After talking to Illumina, they said it might be the library but they cannot exclude a failure of the reagents, so they sent new reagents. I went down from 1000 K/mm² to 700 K/mm² and increased PhiX from 5 % to 12 % and am now running the same library again. This costed me 8 M reads PF (down from 25 M to 17 M). I hope that it'll still be enough for the analysis.

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      • #18
        Originally posted by pmiguel View Post
        Actually that is pretty much what all 600 cycle runs seem to do. Oddly 500-550bp amplicons seems to PANDA merge just fine, so I tended to assume that the instrument was just losing its confidence. But since the phiX error rate also tends to sky rocket at this point, I am not sure what to think.

        --
        Phillip
        This won't work for the amplicon reads, but for the PhiX reads, you can plot the error rates like this:

        bbmap.sh nodisk ref=phix.fa in=reads.fq mhist=mhist.txt qhist=qhist.txt ehist=ehist.txt

        As long as you have at least a few percent of PhiX, it should be somewhat reflective of the error rates by position of all reads. You can use all reads for this, or just the PhiX reads; the results will be similar, since non-PhiX reads won't map to PhiX. However, it will be slightly more accurate using all reads, since that will include reads that are so low quality that Illumina's software does not recognize them as PhiX.
        Last edited by Brian Bushnell; 06-16-2015, 01:49 AM.

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        • #19
          We have been told that the only deleterious effect this observation has is reduced coverage. This appears to jive with observation that there are no general problems with sequence (they seem to overlap fine). This of course does not apply in @ksawatzki's case, since there were N's in reads in that run.

          If you think you are not getting adequate coverage for 600 bp V3 runs, open a ticket with Illumina tech support and contact your local FAS.
          Last edited by GenoMax; 06-16-2015, 05:30 AM.

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          • #20
            Hello everybody,

            The quality drop its a real issue, we already report to Illumina 4-5 Runs, with more or less the same metrics (last one R2=52%Q30). We got cluster density from 900 to 1400 k/mm2 and the quality its the same.
            They admitted that there's a quality issue with these kits (600 cycles), but they still do not know what's the problem. They replaced already 2 cartridges, and we got the same problem than the previous (different lot nr).
            This is not such a big problem for re-sequencing of genomes, since essentially the reads are still usable after more trimming, but for the 16S (V3-V4) samples it is.

            Cheers

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            • #21
              Illumina have advised us not to run 2x300 on these cartridges and to only use them for 2x250 runs. If that doesn't generate enough coverage for our application they will then send replacement reagents.

              Has anyone else recieved this advice?
              Last edited by Bukowski; 06-29-2015, 11:13 PM.

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              • #22
                Same Issues in Buffalo

                We've been running into major issues joining 16s reads for the v1-v3 region. Quality drops like a rock as most of you are also talking about. 2x300 PE MiSeq. They applied the "optical fix" but that did not help.

                They have been reimbursing all of our MiSeq kits, and we were told the same advice - sequencing until you get enough data and they will reimburse. Unfortunately we need the full length 2x300 so that will not work for us.

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                • #23
                  Yeah, it's a shame.

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                  • #24
                    Originally posted by Bukowski View Post
                    Illumina have advised us not to run 2x300 on these cartridges and to only use them for 2x250 runs. If that doesn't generate enough coverage for our application they will then send replacement reagents.

                    Has anyone else recieved this advice?
                    Yes, same thing here

                    Comment


                    • #25
                      The last few Miseq 600 cycles kits we've used have all had rapid increasing error rates after 100-150 cycles of the forward and reverse reads. The FAS's here (Australia) have been alluding to a (global) problem for quite a few months now with the 500 & 600 cycle Miseq kits, but there's been no formal batch flagging from Illumina HQ to date.... Due to the time everyone wastes in re-runs & troubleshooting, in my opinion, this should have happened by now. Sure, they offer to replace kits - but what about the time spent?

                      Mark
                      Last edited by markbvdh; 07-09-2015, 05:14 PM.

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                      • #26
                        We are having the same issues with V3, quality dropping in the forward read even before first 80-100 bases. We tried to get some more information as it seems it is not related to any batch. Has anyone any idea what might be the problem? In our lab we are running lots of targeted DNA sequencing, even using same library preps and MASTRs and we do see decline in quality since the middle of this summer and it affects different LOTs. As a response we get replacement kits, but yeah that does not solve the issue of wasting time and producing bad data.

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                        • #27
                          Low %>Q30 on R2 for 2x300 libraries

                          We have had numerous quality complaints from our customers performing 2x300 and 2x250 MiSeq runs (predominately amplicon libraries) where the %>Q30 is approximately 60% or less due to the “poor” quality of Read 2, and in some instances a significant number of clusters do not pass filter.

                          In a handful of cases, Illumina tech support cited issues after reviewing SAV, e.g.,
                          • "There may have been a transient fluidics issue that occurred at the beginning of the run"
                          • "Loss of focus at the end of the run likely caused by over clustering"
                          • "Quality drop corresponds with an increase of A base calling. This is indicative of running out of insert to sequence."

                          Illumina replaced several kits and dispatched a field technician to adjust the MiSeq due to a non-optimal Z-focus setting. Even after this adjustment, we are still seeing low PF rates for our 2x300 and 2x250 runs, and tech support states that our MiSeq is operating within spec. The only suggestions offered by Illumina are that the samples run, e.g., amplicon libraries, are low in base diversity and that we should continue to decrease loading concentration and increase PhiX spike-in, which we already do and does not appear to significantly improve the situation.

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                          • #28
                            You can calculate the insert size distribution with BBMerge like this:

                            bbmerge.sh in=r1.fq in2=r2.fq ihist=ihist.txt vloose qtrim2=r


                            (those settings are designed for low-quality reads).

                            This will give you an indication of what fraction of your inserts are shorter than read length, and thus whether the third bullet point is a plausible explanation.

                            Also, can you post a base frequency histogram (by position)? And, how much PhiX are you using; are you multiplexing different amplicons together, or is there just one kind in a lane; and are you using variably-sized sequencing primers to stagger the read starts?
                            Last edited by Brian Bushnell; 09-17-2015, 10:23 AM.

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                            • #29
                              Modern MiSeq Control Software doesn't seem to benefit from higher phiX content -- beyond ~5-10% necessary for the instrument focus on. But dropping the clustering concentration down enough does seem to help. Obviously you can then end up with runs that are below Illumina's specifications for a given MiSeq run.

                              Given the amount of trouble they are having with this, I think they should have separate specs for low-plex (eg, single-plex) amplicon runs in their sales advertisements. That way people know they are not going to be able to push the system as much when doing amplicon work.

                              --
                              Phillip

                              Comment


                              • #30
                                Originally posted by pmiguel View Post
                                Given the amount of trouble they are having with this, I think they should have separate specs for low-plex (eg, single-plex) amplicon runs in their sales advertisements. That way people know they are not going to be able to push the system as much when doing amplicon work.
                                This point deserves repeating Phillip. Managing client expectations during initial consultations for MiSeq amplicon projects is huge. I generally advise them to plan their experiments with the expectation of 9-10 million usable read pairs from a MiSeq v2 PE250 run for amplicon libraries. I explain that for amplicon libraries we lower the cluster density; that the PhiX control library will take up 5-10% of the reads and with large, complex index sets the fraction of reads with non-assignable indexes is higher.

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