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Old 07-06-2010, 04:15 PM   #1
DoubleA
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Default 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?

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Double A
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Old 07-09-2010, 08:40 AM   #2
adamdeluca
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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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Old 07-09-2010, 08:52 AM   #3
DoubleA
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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.

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DoubleA
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Old 07-09-2010, 09:24 AM   #4
adamdeluca
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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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Old 07-09-2010, 10:01 AM   #5
DoubleA
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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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Old 07-09-2010, 11:54 AM   #6
NextGenSeq
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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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Old 11-14-2010, 08:40 PM   #7
KevinLam
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Quote:
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?
http://www.genomics.agilent.com/Gene...etail&PageID=4

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 at 08:41 PM. Reason: added info
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