attempting to sequence human MHC region

DoubleA · 07-06-2010, 04:15 PM

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

adamdeluca · 07-09-2010, 08:40 AM

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.

DoubleA · 07-09-2010, 08:52 AM

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

adamdeluca · 07-09-2010, 09:24 AM

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.

DoubleA · 07-09-2010, 10:01 AM

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

NextGenSeq · 07-09-2010, 11:54 AM

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.

KevinLam · 11-14-2010, 08:40 PM

Quote:

Originally Posted by NextGenSeq

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.

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07-06-2010, 04:15 PM	#1
DoubleA Member Location: Los Angeles Join Date: Jul 2010 Posts: 16	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

07-09-2010, 08:40 AM	#2
adamdeluca Member Location: Iowa City, IA Join Date: Jul 2010 Posts: 95	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.

07-09-2010, 08:52 AM	#3
DoubleA Member Location: Los Angeles Join Date: Jul 2010 Posts: 16	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

07-09-2010, 09:24 AM	#4
adamdeluca Member Location: Iowa City, IA Join Date: Jul 2010 Posts: 95	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.

07-09-2010, 10:01 AM	#5
DoubleA Member Location: Los Angeles Join Date: Jul 2010 Posts: 16	Wow! I just checked the bed file and you are correct. Many important genes do not have any baits. Thanks again for your help!

07-09-2010, 11:54 AM	#6
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	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.