Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • ATAC-seq plasmid contamination after transfection

    HI all

    I am trying to produce ATAC-seq libraries from transfected cells, but it seems a pain...

    I have produced good libraries from non-transfected cells (sequencing was OK), but when I transfect cells with a plasmid I get, of course, a lot of tagmentation in the plasmid, such that most of my reads are plasmid-derived. I would like not to diminish the plasmid concentration, because all my experiment so far have been done using the same conditions.

    We are trying to solve this problem but it does not look easy...

    My idea is to get rid of the plasmid before tagmentation and probably use AMPure beads to suck it up just after lysing the cells. But I am not sure how this will work because there may be complications (I am going to test this next week, I think)

    Another possibility is to check the tagmented genome (before amplification) on a gel and see if it is be possible to separate the plasmid from the genome. This actually raises the question if the genomic DNA is fragmented after tagmentation (I think it is, unfortunately... can you confirm?)

    We were also considering sequence capture to get rid of the plasmid fragments, but this also has some problem in the fact that the adapters may then hybridise together resulting in non specific sequence capture.

    Please let me know if you have found such a problem and came up with a solution.
    Very much appreciated

    Bests
    Dam

  • #2
    This isn't something that I have tried but I have thought about it. If your plasmid isn't too long you could possibly try using long biotinylated oligos that will hybridize to known sequence in your plasmid. You then pull down plasmid DNA fragments from your ATACseq sample post-tagmentation, pre-amplification.

    I would be interested to hear other people's thoughts about the viability of such a method.

    Also there is another method which is likely to be more viable since it has already been published. I think there are some papers out using CRISPR/Cas9 and sgRNAs to deplete mitochondrial DNA in ATACseq samples. I don't see why that can't be applied to deplete your plasmid DNA.

    check out this link

    http://biorxiv.org/content/early/2016/11/22/087890

    Also, to answer your question, the DNA is fragmented after tagmentation.

    Comment


    • #3
      It might be possible to adapt NuGEN AnyDeplete technology to get rid of plasmid sequences.

      Sequence capture will not work as only one strand of plasmid read will be captured and the other strand will stay in supernatant along the host fragments which will be amplified or sequenced. If you use host probes for capture it might be expensive as probes have to be synthesised for whole host genome.

      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, Yesterday, 06:37 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      49 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      66 views
      0 likes
      Last Post seqadmin  
      Working...
      X