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  • Poor quality 2nd end from paired-end sequencing on HiSeq

    We are seeing very low quality for the first few bases of the 2nd end in paired-end sequencing on the HiSeq. We have data from 6-7 experiments where the library prep and sequencing was conducted at 3 different sequencing centers around the country. The library preps varied between centers, but each center did the sequencing on HiSeq 2000s. It seems like there is a problem with chemistry on the sequencer.

    We were told by one sequencing center:
    "It turned out that there was a NaOH problem…the protocol uses NaOH to strip of the index prior to sequencing the 2nd read. While the NaOH reagent sat on the machine, for some reason, it degraded and proper removal of the index was not achieved. Adding fresh NaOH a day before index2 did the trick. The Qscore looks amazing...."

    Another sequencing center said:
    "They [Illumina] have suggested that NaOH is not working well anymore to denature index1 away. If this is not complete, it will continue sequencing 7 dark cycles after index1 and then 8nt from the adapter, which is exactly the adapter sequence that is the top match in my 8mer analysis."

    I'm attaching an image of what our poor quality scores look like for the 2nd end; the first end looks great. I'd like to hear if anyone else is seeing the same problem and whether you have a similar or different answer from Illumina or your sequencing centers.

    Apologies if you're seeing this post a second time. One Senior Member of SeqAnswers suggested that I post this as a new thread. I had previously attached it to a thread about a similar problem on the miSeq, which could very well be due to a different problem.
    Attached Files

  • #2
    Originally posted by agent99 View Post
    We are seeing very low quality for the first few bases of the 2nd end in paired-end sequencing on the HiSeq. We have data from 6-7 experiments where the library prep and sequencing was conducted at 3 different sequencing centers around the country. The library preps varied between centers, but each center did the sequencing on HiSeq 2000s. It seems like there is a problem with chemistry on the sequencer.

    We were told by one sequencing center:
    "It turned out that there was a NaOH problem…the protocol uses NaOH to strip of the index prior to sequencing the 2nd read. While the NaOH reagent sat on the machine, for some reason, it degraded and proper removal of the index was not achieved. Adding fresh NaOH a day before index2 did the trick. The Qscore looks amazing...."

    Another sequencing center said:
    "They [Illumina] have suggested that NaOH is not working well anymore to denature index1 away. If this is not complete, it will continue sequencing 7 dark cycles after index1 and then 8nt from the adapter, which is exactly the adapter sequence that is the top match in my 8mer analysis."

    I'm attaching an image of what our poor quality scores look like for the 2nd end; the first end looks great. I'd like to hear if anyone else is seeing the same problem and whether you have a similar or different answer from Illumina or your sequencing centers.

    Apologies if you're seeing this post a second time. One Senior Member of SeqAnswers suggested that I post this as a new thread. I had previously attached it to a thread about a similar problem on the miSeq, which could very well be due to a different problem.
    I agree with HeinKey, the explanation you were given makes no sense. If NaOH had failed to remove the product strands from read1 and the index read it might have interfered with synthesis of the new template strand, but that would have been detrimental to the quality of the entire reverse read, not just a base or two.

    Also, isn't the read1 template removed with NaOH prior to annealing the index primer and initiating the index read? If your index read was good, seems like that is an indication that your NaOH was good at that point. How did it go bad in the intervening time before the reverse read?

    To me it looks like something bad happened during cycle2 of the reverse read. But something that would wipe out the quality of all the tiles but then allow the rest of the read to proceed unabated can't be chemistry.

    My guess would be a communication error that caused the scan focus to use the wrong offsets for cycle2 of that read. The problem then corrected itself during initiation of scanning of cycle3. But the software would still discount the quality values of the cycle following a low quality cycle.

    As to why, if I understand correctly, several batches of your samples got hit by this even though they were sent to several different facilities -- well hard to say. Maybe they all upgraded to a new version of HCS with this bug at around the same time? Illumina then decides the NaOH was to blame, replaces the run reagents and tells people to make sure to add fresh NaOH to their instruments 1 day prior to turnaround. Then, silently, the Illumina informatics guys push out a new version of HCS not subject to this issue which fixes it? Pure speculation on my part, of course. But we have been seeing communication error-based glitches with some recent versions of HCS. Not exactly the same as this one though. But they look similar in some aspects.

    --
    Phillip

    Comment


    • #3
      Originally posted by pmiguel View Post
      Then, silently, the Illumina informatics guys push out a new version of HCS not subject to this issue which fixes it? Pure speculation on my part, of course.
      The only way they could sneak that new version on our machines though is if the service engineer installs it

      Originally posted by pmiguel View Post
      But we have been seeing communication error-based glitches with some recent versions of HCS. Not exactly the same as this one though. But they look similar in some aspects.

      --
      Phillip
      The "communication errors" are hard to pin down since they do not occur with predictability. We have been running a new HCS version in beta on a test HiSeq 2000 and these sometimes occur there as well.

      It is baffling that samples sent to three separate facilities had the same issue. That may indicate some characteristic of the libraries (though possibility of HCS having a very specific bug can't be eliminated).

      Comment


      • #4
        We had recently seen this sort of pattern for read 2 (big dip in quality at beginning, though in our case it was at cycle 9 and then decay over the remainder of the run) with some 2-D MiSeq paired-end runs.

        Those samples were known to be "not standard". We are investigating the issue with help of Illumina tech support.

        Comment


        • #5
          Originally posted by GenoMax View Post
          The only way they could sneak that new version on our machines though is if the service engineer installs it
          Then if you never saw that particular issue on your instruments, that may be why.
          Originally posted by GenoMax View Post
          The "communication errors" are hard to pin down since they do not occur with predictability. We have been running a new HCS version in beta on a test HiSeq 2000 and these sometimes occur there as well.

          It is baffling that samples sent to three separate facilities had the same issue. That may indicate some characteristic of the libraries (though possibility of HCS having a very specific bug can't be eliminated).
          The OP made it sound like the libraries were being constructed in different places as well.

          --
          Phillip

          Comment


          • #6
            Originally posted by pmiguel View Post
            Then if you never saw that particular issue on your instruments, that may be why.
            Does that mean Illumina pushes new versions of HCS over the net to your machine if it is directly connected to internet? I did not know that. I am glad our instruments are isolated from network.

            Originally posted by pmiguel View Post
            The OP made it sound like the libraries were being constructed in different places as well.
            I should have said sample characteristics. If the libraries were made at three places then they have been made consistently.

            Comment


            • #7
              Originally posted by GenoMax View Post
              Does that mean Illumina pushes new versions of HCS over the net to your machine if it is directly connected to internet? I did not know that. I am glad our instruments are isolated from network.
              No. MiSeqs are close to that -- you just push one button from inside MCS and the update happens.
              But some would just update any time they saw there was a new HCS available. I usually do, but not always.
              Originally posted by GenoMax View Post
              I should have said sample characteristics. If the libraries were made at three places then they have been made consistently.
              I would be at a lost to think of a sample characteristic that would give very low quality at the 2nd base of the reverse read -- outside of an amplicon, maybe.

              --
              Phillip

              Comment

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