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  • Very low average % >= Q30

    Dear all,

    I need an advice regarding latest NextSeq 500 sequencing run. Given - NextSeq 500, HighOutput 75 cycles sequencing kit v2, highly multiplexes (192 libraries) sequencing pool of genomic DNA library samples prepared with in-house tagmentation procotol and seven additional Cut&Tag libraries. All libraries have 8bp i5 and i7 barcodes. Bioanalyzier profiles and library concentrations looks fine as usual. The DNA libraries are routine for our lab and never caused such kind of troubles before. Cut&Tag libraries are rather new test samples. I do not know why they would affect the sequencing, but they might be considered as an unknown variable in the equation.

    The library pools were diluted to 2nM final concentration and denatured according standard Illumina instructions. The cluster density is about 200, which is quite typical for this machine and our sample preparation, however only 1.91% of clusters passed filter per lane giving 0.92Gb yeild. What are the first things to check? Thanks a lot in advance.

    PS One of the components of the kit expired about two months ago, so I can't really contact Illumina, but this was never an issue in the past. Most sequencing kits works well even years after the expiration date.

  • #2
    Malfet that's very strange. My immediate suspicion would be the Cut&Tag libraries that were included. Have you run them on another instrument before? I've had some issues in the past when I mixed together libraries and the quality and yield dropped considerably. In your case, it seems a bit extreme, but I can't think of anything else.

    And I agree with you, I've used expired reagents plenty of times and there was no issue. It would really only cause an issue if it had been thawed and then refrozen.

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    • #3
      Originally posted by Ben3 View Post
      Have you run them on another instrument before?
      Hi, nope, it's the first time. On Bioanalyzer Cut&Tag libraries also looked fine, except may be one that did not show the expected distribution, but no primer-dimers in high amounts or anything like that. I will probably try to exclude Cut&Tag libraries first and see what will happen, because I do not have other ideas at the moment...

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      • #4
        Malfet well, I've honestly never had good luck when I was combining libraries unless I did a nano run on a MiSeq to balance them out. Hopefully it looks better when you run it again without the Cut&Tag libraries. Let me know if it looks better and good luck!

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