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  • Strange peaks in TruSeq RNA library

    Hi all
    I've run these TruSeq RNA libraries I received on the Bioanalyser HS Kit and they appear to be have strange peaks, almost as if there was a ladder contamination (although the peaks don't correlate with the HS ladder sizes). I've run these samples twice now, preparing fresh gel-dye and using a new tube of marker, but I get the same traces.
    Has anyone got any explanations?
    Attached Files

  • #2
    Hi Tony,
    Just a guess, but:
    (1) A MW ladder, as you mention as a possibility, was somehow introduced at some point.

    (2) It could be highly expressed transcripts in your sample. They might not be visible in the original total RNA QC because the

    (3) Horror of horrors, the TruSeq kit includes some crazy control fragments that are supposed to help you trouble shoot/QC your libraries down stream. I would as soon jab a pippeter in my eye as add extraneous DNA to samples during library construction. But it is part of the standard TruSeq protocol...

    Here are their sizes according to the "Illumina Adapter Sequence Letter" you can request from your Illumina FAS:

    CTE2 - 150bp
    CTE2 - 250bp
    CTE2 - 350bp
    CTE2 - 450bp
    CTE2 - 550bp
    CTE2 - 650bp
    CTE2 - 750bp
    CTE2 - 850bp
    CTE1 - 123bp
    CTE1 - 223bp
    CTE1 - 323bp
    CTE1 - 423bp
    CTE1 - 523bp
    CTE1 - 623bp
    CTE1 - 723bp
    CTE1 - 823bp
    CTA - 150bp
    CTA - 250bp
    CTA - 350bp
    CTA - 450bp
    CTA - 550bp
    CTA - 650bp
    CTA - 750bp
    CTA - 850bp
    CTL - 150bp
    CTL - 250bp
    CTL - 350bp
    CTL - 450bp
    CTL - 550bp
    CTL - 650bp
    CTL - 750bp
    CTL - 850bp

    Then, I guess, there would be adapter ligated on to some/all of them? The universal strand is 58 nt and the index strand is 63. After enrichment PCR (if any) the frags would be 121 bp longer?

    That would give you these size frags:

    244
    271
    344
    371
    444
    471
    544
    571
    644
    671
    744
    771
    844
    871
    944
    971

    Some of those might explain some of your extra peaks. The 128 bp and 3109 bp peaks do not seem to fit that hypothesis though. 128 is probably an adapter dimer, though. Maybe a concatamer of some of the control frags?

    --
    Phillip

    Comment


    • #3
      Could be from the Ampure beads remaining in solution? I tend to leave the plate on the magnetic stand for a few minutes more than in the manual (7 vs. 5 minutes). Also check pipet tip carefully to see if any beads remain. Can be hard to take supernatant without taking up some beads. Also are you seeing this in all libraries?

      By the way are you doing the standard PCR enrichment (all 15 cycles)? Not seeing the ubiquitous 'bump' ~600-2000bp, wondering why.

      Comment


      • #4
        any updates on this? we started to see something similar..

        Comment


        • #5
          Originally posted by pmiguel View Post
          I would as soon jab a pippeter in my eye as add extraneous DNA to samples during library construction. But it is part of the standard TruSeq protocol...
          ha ha.

          Totally agree. Was trying to explain to a student collaborator why we were leaving out the 'in-line controls'. He didn't like my first answer so I came back with 'because it's the stupidest #$%@@$ idea I've ever heard of'.

          Anyway, agree my guess would be too much of the in-line controls were added.
          --------------
          Ethan

          Comment


          • #6
            We saw the same peaks but a very lower extent in some Truseq DNA libraries. Sequencing of this libraries confirmed that those peaks are truseq controls. After that, we never use again controls.

            Comment

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