Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Calculating capture efficiency

    Hi all,

    I want to calculate what percentage of my sequenced custom capture is actually off-target and I can't get my head around how to do this - does anyone have any tips?

    Many thanks.

  • #2
    A couple of ways you can do this.

    1. The easiest would be to create a reference sequence for your custom capture, and then align your data to the whole genome and your custom reference sequence (give your custom reference sequence 500 bp flanking region). Then see how many reads align to the whole genome but don't align to your custom capture. There may be a bit of ambiguity due to some reads aligning to the custom capture that don't align to the whole genome if they have high homology to multiple portions of the human genome, but this won't be a big issue from my experience.

    2. If you have aligned to the whole genome and don't want to go through the trouble aligning to the custom reference sequence, you can run GATK depth of coverage for the whole genome and also for the specific targeted intervals (again, + and - 500bp flanking regions). Then compare the total amount of coverage.

    Comment


    • #3
      Try Picard (http://picard.sourceforge.net/comman...ulateHsMetrics)

      Comment


      • #4
        I use BEDTools. Get a .bed file of your capture regions. Align to the whole genome (which is in general a better idea than just aligning to target region), then intersect your whole genome .bam with the .bed file. You might pad the bed file first, though if you use the exact coordiantes, you will not lose reads that hang off your target region.

        Then count (with samtools flagstat, or something like that) how many lines are in your bam before and after filtering.

        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