Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • RRBS library long fragments

    Dear All,

    I just made a RRBS library. The problem is that I have a lot of long fragments that is not really good since they probably do not contain CpG rich areas.

    This is the profile of the library:


    Can anyone help me with what I should do?

    It is made from 16 samples pooled together. DNA is from blood with good quality. Msp1 digestion worked.

    Please help. Do you believe that i can rescue the library with size selection?

    Cheers
    Emil

  • #2
    The short fragments looks typical of human or mouse RRBS but I think that PCR extension time has been excessive resulting in large fragments amplification as well or PCR has been overcycled. It is OK to sequence it without any size selection because clustering will favour short fragments so a small number of reads will be from large fragments. You might need to increase sequencing depth in comparison to a size selected library for optimum coverage.

    If large fragments are result of MspI digestion (not degraded DNA that has been end repaired and adapted) then they will have at least one CpG site. The only issue is that you probably will not get enough sequencing coverage for those.

    Comment


    • #3
      The markers for this BA trace are also being called incorrectly, so you will want to go back and manually set them in order to get an accurate size distribution. That upper peak should be at 10380bp, but it is showing up at ~800. This will cause the software to underestimate the fragment sizes.
      Josh Kinman

      Comment


      • #4
        Thank you very much both of you. The samples have now been sequenced. And I will test the analysis in not too long.

        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
        11 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-22-2024, 10:03 AM
        0 responses
        51 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-21-2024, 07:32 AM
        0 responses
        67 views
        0 likes
        Last Post seqadmin  
        Working...
        X