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  • attempting to sequence human MHC region

    I wanted to know if anyone has experience sequencing the human MHC region (~4 Mb). My plan is capture the region using a custom Agilent SureSelect kit (exons, intron, 5' and 3' UTRs). After masking the repetitive regions, I found that ~30,000 baits will cover the remaining 2 Mb of sequence at 1X tiling. We would like to generate libraries for both the 454 and GAIIX machines.

    Our next-generation core facility beta-tested Agilent's Human All-Exon SureSelect kit with good results except for the MHC region. Unfortunately, ~70% of the genes in this region were lacking sequence in one or more exons. I'm getting the feeling that it is probably impossible to sequence this region using a hybridization sequence capture approach. Any thoughts?

    Thanks,
    Double A

  • #2
    The Agilent All Exon kit only targets ~80% of coding refseq exons.
    Really quick numbers here: there are 560 baits in this kit that target the MHC region and ~1300 exons. The kit just doesn't target the region very well, your custom design will likely work fine.

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    • #3
      Thanks for the information! That's really surprising. I think my 55000 baits will do a better job than their 560! How did you find out only 560 baits target the MHC region? Did you find a "BaitTiling_bed" file for Agilent's Human All-Exon SureSelect kit? I would really like to look at their bait layout on Santa Cruz's genome browser.

      Thanks,
      DoubleA

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      • #4
        You can get a bed file of the baits from the Agilents eArray site. I used bedTools to extract the baits targeting the MHC region. You could also upload the bed file from earray to UCSC as a custom track.

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        • #5
          Wow! I just checked the bed file and you are correct. Many important genes do not have any baits. Thanks again for your help!

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          • #6
            Note Agilent is in the process of updating their "whole exome" enrichment kit. Apparently, a lot of people have complained at the large number of missed exons.

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            • #7
              Originally posted by NextGenSeq View Post
              Note Agilent is in the process of updating their "whole exome" enrichment kit. Apparently, a lot of people have complained at the large number of missed exons.
              by updates did you mean the new 50Mb kit?
              Explore Agilent’s applications and solutions for your genomics lab. Discover tools for next-generation sequencing (NGS), microarrays, CRISPR, PCR/qPCR, sample quality control (QC), and data analysis platforms with Agilent's full genomics lab solutions.


              Just Launched:
              Agilent SureSelect Human All Exon 50Mb Kit; Provides the most complete definition of the human exome on the market!
              8/4/2010

              SANTA CLARA, Calif. And CAMBRIDGE, England, Aug. 4, 2010 –

              Agilent Technologies Inc. (NYSE: A) today introduced the SureSelect Human All Exon 50 Mb Target Enrichment kit for next-generation DNA sequencing. The kit enables researchers to streamline experiments by sequencing the expressed genome while discarding regions that are not of interest.
              Last edited by KevinLam; 11-14-2010, 09:41 PM. Reason: added info
              http://kevin-gattaca.blogspot.com/

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