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#1 |
Member
Location: Los Angeles Join Date: Jul 2010
Posts: 16
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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 |
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#2 |
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Location: Iowa City, IA Join Date: Jul 2010
Posts: 95
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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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#3 |
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Location: Los Angeles Join Date: Jul 2010
Posts: 16
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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.
Thanks, DoubleA |
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#4 |
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Location: Iowa City, IA Join Date: Jul 2010
Posts: 95
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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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#5 |
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Location: Los Angeles Join Date: Jul 2010
Posts: 16
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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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#6 |
Senior Member
Location: USA Join Date: Apr 2009
Posts: 482
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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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#7 | |
Senior Member
Location: SEA Join Date: Nov 2009
Posts: 197
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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.
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http://kevin-gattaca.blogspot.com/ Last edited by KevinLam; 11-14-2010 at 08:41 PM. Reason: added info |
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