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  • #16
    Another run. Not as bad this time. Still not 2500-level good (for read 2) but not as terrible as before.

    A few things we've learned thus far (empirically and from talking Illumina) regarding the 3k:

    1) Anything short will take over the ordered flowcell with a vengeance. As best you can, DO NOT have library fragments <300bp (150bp insert). ALL adapter dimer must be gone. Do at least a 0.8x SPRI cleanup. This flowcell is much less forgiving than the 2500.

    2) If you have libraries on the shorter side (200-300bp insert) it is better to OVER load than UNDER load.
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    • #17
      Perhaps this is the reason why HiSeq X was such a controlled release and for a specific application. Patterned flowcells generate a lot of data but the quality is good (in terms of duplicates) only when libraries fit a narrow/strict quality profile.

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      • #18
        Timely article from James Hadfield reiterating some of the points from GW_OK

        The HiSeq 4000 was Illumina's way of making the patterned flowcell technology available to non X Ten customers, and opening up patterned ...

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        • #19
          Thanks for the link. The article does not present anything new (yet)- James just received his HS4000.
          We have been very happy with it, the exception being that the qualities of the PE150 reads are no longer as good as they were last spring and summer. The other mentioned caveats can be dealt with; long insert libraries need to be run on the Hiseq 2500 of course.

          Originally posted by Bukowski View Post
          Timely article from James Hadfield reiterating some of the points from GW_OK

          http://core-genomics.blogspot.co.uk/...d-to-know.html

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          • #20
            Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad, especially in read 2, only 60% and 70% of bases >Q30, which doesn't pass the Illumina specs. In this case, is it a common practice for the facility to redo the sequencing for us? We ask because it sounded like they don't want to redo it for us... How about the policy at your sequencing core?

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            • #21
              Originally posted by melop View Post
              Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad, especially in read 2, only 60% and 70% of bases >Q30, which doesn't pass the Illumina specs. In this case, is it a common practice for the facility to redo the sequencing for us? We ask because it sounded like they don't want to redo it for us... How about the policy at your sequencing core?
              Local policies at facilities differ and it is possible that your facility may not have a specific one for 4000 data due to the peculiarities you have been reading about in this thread.

              That said, there is certainly enough reason to show your QC data to the facility and discuss your concerns/available options. If only your samples show this trend and other samples on the flowcell did not have a problem then that may be a reason why the facility may seem reluctant to redo your samples.

              There is no harm (except a bit of additional work) for the facility to open a ticket with Illumina and have them look at the metrics of run in question. If Illumina provides replacement reagents (that is they determine that there is potential reagent/hardware issue) then you should be able to get your samples re-sequenced at no charge. If no free replacement kit is forthcoming then your options revert to getting the sample sequenced on a different sequencer (2500) at full and/or subsidized cost (since the facility made your libraries they have a responsibility to get you usable data).
              Last edited by GenoMax; 01-19-2016, 03:07 PM.

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              • #22
                I see. But is that a common practice to let the end user bare the loss even if the facility prepares the library knowing that they are going on a HiSeq 4000? I understand that if Illumina agrees to replace the reagents then there will be no problem, but I will be surprised if that should become the end user's burden if such a performance issue is caused by inadequate library prep (say insert size not tight enough or negligence in adapter cleaning, suggested by previous posts). One problem is that this core does not have any written policy regarding quality, and their policies all seem to be post hoc .....

                Originally posted by GenoMax View Post
                Local policies at facilities differ and it is possible that your facility may not have a specific one for 4000 data due to the peculiarities you have been reading about in this thread.

                That said, there is certainly enough reason to show your QC data to the facility and discuss your concerns/available options. If only your samples show this trend and other samples on the flowcell did not have a problem then that may be a reason why the facility may seem reluctant to redo your samples. There is no harm (except a bit of additional work) for the facility to open a ticket with Illumina and have them look at the metrics of run in question.

                If Illumina provides replacement reagents (if they determine that there is potential reagent issue) then you should be able to get your samples re-sequenced at no charge. If no free replacement kit is forthcoming then your options revert to getting the sample sequenced on a different sequencer (2500) at your cost.

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                • #23
                  Originally posted by melop View Post
                  I see. But is that a common practice to let the end user bare the loss even if the facility prepares the library knowing that they are going on a HiSeq 4000? I understand that if Illumina agrees to replace the reagents then there will be no problem, but I will be surprised if that should become the end user's burden if such a performance issue is caused by inadequate library prep (say insert size not tight enough or negligence in adapter cleaning, suggested by previous posts). One problem is that this core does not have any written policy regarding quality, and their policies all seem to be post hoc .....
                  You are referring to two separate issues and they need to be addressed separately. Sounds like you are ok with the run metrics explanation.

                  If you have questions about the quality of the libraries that were prepared then ask the facility to share their library QC data with you. Ask them if they bead treated the libraries as recommended? If the libraries look suboptimal then they may need to clean them up and re-run your samples at no cost. Again things you should discuss in good faith with your facility.

                  There are current instances (e.g. MiSeq 2x300 kits) where dismal Q-scores seem to rule late in read 2 but that does not affect the actual sequence. That may apply in your case as well. You may have perfectly good sequence data that has a Q-score issue that people have been discussing in this thread.
                  Last edited by GenoMax; 01-19-2016, 03:35 PM.

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                  • #24
                    I encourage you to measure the quality-score accuracy of the runs, as per this thread (near the top of the first post), if you want hard data to show to your sequencing facility. As GenoMax mentioned, quality scores do not always correctly reflect accuracy - I've seen them both too high and too low.

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                    • #25
                      It is possible (if the library was correctly sized and the run was not overloaded; unfortunately the latter is hard to diagnose on the HS4000 ) that the problem is due to the reagents as discussed in this thread. If we spend some time with the Illumina tech support on the phone, Illumina will usually replace the reagents for us in these cases.

                      We would definitely re-run your libraries (as they were generated by the core), but is actually very difficult to assign responsibility/blame for failures on the HS4000 if only a single lane is run.


                      Originally posted by melop View Post
                      Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad, especially in read 2, only 60% and 70% of bases >Q30, which doesn't pass the Illumina specs. In this case, is it a common practice for the facility to redo the sequencing for us? We ask because it sounded like they don't want to redo it for us... How about the policy at your sequencing core?

                      Comment


                      • #26
                        Originally posted by luc View Post
                        It is possible (if the library was correctly sized and the run was not overloaded; unfortunately the latter is hard to diagnose on the HS4000 ) that the problem is due to the reagents as discussed in this thread. If we spend some time with the Illumina tech support on the phone, Illumina will usually replace the reagents for us in these cases.

                        We would definitely re-run your libraries (as they were generated by the core), but is actually very difficult to assign responsibility/blame for failures on the HS4000 if only a single lane is run.
                        I completely agree.

                        I am puzzled, though, why any Core would not immediately have had the run replaced by Illumina and reloaded if it did not meet spec.

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                        • #27
                          Just to add another voice, we noticed this on our validation runs at install in late Nov/December. Q30s dropping after 100 cycles of read 2, ending the run at ~40%. Error rates up above 7-8% as well. It was actually most obvious in the control lanes of PhiX that we ran. Seems like it must be a reagent issue, probably with enzyme or cleavage mix.
                          This thread just prompted me to email our FAS about it again. We didn't have a very satisfactory response from tech support, they said they hadn't seen enough 4K runs to determine what was "normal", but that our runs meet spec.
                          My PI said he's heard through the grapevine that this problem is widespread though - at Yale, Cornell, NYGC etc.
                          Last edited by neurula; 01-26-2016, 09:22 AM.

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                          • #28
                            Originally posted by Brian Bushnell View Post
                            I encourage you to measure the quality-score accuracy of the runs, as per this thread (near the top of the first post), if you want hard data to show to your sequencing facility. As GenoMax mentioned, quality scores do not always correctly reflect accuracy - I've seen them both too high and too low.
                            This looks interesting and definitely will be useful for downstream analyses.
                            However I feel that the statistics should be measured in the same way Illumina does, since Illumina guarantees 75% bases >Q30 based on their software output.

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                            • #29
                              Originally posted by luc View Post
                              It is possible (if the library was correctly sized and the run was not overloaded; unfortunately the latter is hard to diagnose on the HS4000 ) that the problem is due to the reagents as discussed in this thread. If we spend some time with the Illumina tech support on the phone, Illumina will usually replace the reagents for us in these cases.

                              We would definitely re-run your libraries (as they were generated by the core), but is actually very difficult to assign responsibility/blame for failures on the HS4000 if only a single lane is run.
                              So I have 2 lanes, both of which fall below 75% >Q30 (60% and 70%). As far as I know, the other 6 lanes are all borderline, right at 75% >Q30. So the average across the whole flowcell would still be below standard. So perhaps there's a combination of different problems. I'm not sure how successful will they be in negotiating with Illumina with this borderline case though.

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                              • #30
                                Originally posted by GW_OK View Post
                                I completely agree.

                                I am puzzled, though, why any Core would not immediately have had the run replaced by Illumina and reloaded if it did not meet spec.
                                We are puzzled too. Last time I heard from them the core told us that they will only replace the run if Illumina replaces reagents for them. They were vague about what to do if Illumina doesn't. That was why I asked to see if this is a common practice among the other cores.

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