Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Deep Seq Barcode 'crossover'

    Hello, we have just completed a run with 17 barcodes in a single E120. Three samples have deep coverage for one set of genomic fragments, 14 have deep sequence for a different set of fragments. Where depth gets up to and over 5,000x we start to see that fragment pop up in all other samples, we're looking at a 0.002 - 0.003% cross over.

    Does anyone see anything similar in their high coverage samples?..perhaps you haven't noticed it?

    Lab contamination was our first thought (and we're not ruling that out yet); however, as there were 17 samples, one of the barcodes (#17) came from an unopened kit, we had never used #17 before. Also the two sets of fragment libraries were created weeks apart so I could accept the first set contaminating the second I can't see how both could contaminate each other.

    If this were random mis-calling of barcodes or random sequnce error then we would see the 'cross over' to barcodes that were not used ......and we don't.

    Could there be any sort of mix up going on in the emPCR? carried over barcodes?..... What about in software? could there be a binning problem with high coverage fragments?

    JPC

  • #2
    A correction from my last post. The cross over is apparent in barcodes beyond the 17 that we have used, so perhaps we are seeing mis-calling or a barcode binning problem?

    Comment


    • #3
      what level of mismatch tolerance do you have set for barcode binning?

      Comment


      • #4
        Hi snetmcom, thanks for your input.

        The value is set to 0, this is not something we have ever changed and in fact this if the first I have heard of it!

        JPC

        Comment


        • #5
          I wouldn't change that mismatch tolerance value in a hurry. 0 sounds like a good idea.

          Thanks for the warning JPC.

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Strategies for Sequencing Challenging Samples
            by seqadmin


            Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
            03-22-2024, 06:39 AM
          • seqadmin
            Techniques and Challenges in Conservation Genomics
            by seqadmin



            The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

            Avian Conservation
            Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
            03-08-2024, 10:41 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, 03-27-2024, 06:37 PM
          0 responses
          12 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-27-2024, 06:07 PM
          0 responses
          11 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          53 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          68 views
          0 likes
          Last Post seqadmin  
          Working...
          X