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Old 12-19-2011, 11:31 AM   #1
Marius
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Default Targeted sequencing of large genomic fagments (100 kb) with NGS

Hi everyone,

I'm looking for a protocol that describes how to sequence in many individuals a specific stretch of DNA of about 100 kb.

My idea is to PCR amplify this genomic sequence by performing simple, mutliple PCRs with ovelapping products. Since I have a reference genome, primer design won't be any problem. Then, I would simply shear these PCR products to the appropriate fragment size (depending on the platform) and ligate an adapter to them. This I would do seperately for every individual specimen. In case of a HiSeq Illumina platform, I would shear individual PCR products to a length of about 100 bp and ligate an adapter to them that holds the Illumina primer site and a specific barcode. Afterwards I would pool these sheared-barcode-ligated PCR products of many individuals and sequence on a HiSeq.
Once I get the data, I could then make alignments for every individual (the individual specimen can be inferred by looking at the barcode), which I guess shouldn't have any gaps if I have enough reads per indidivuals available, since reads will overlap (by chance event du to rndom shearing of PCR products).

This is just how I would do it.

Now, PLEASE, I'm looking for any protocols that tell me how to sequence such long-stretch DNA from mutliple individuals on a NGS machine. Can be Roche 454 or Illumina or any other NGS platform. It can be anything like I tried to describe above or a completely different approach.
Thank you for ideas, protocols, relevant papers etc.!

Last edited by Marius; 12-19-2011 at 11:38 AM.
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Old 12-19-2011, 03:27 PM   #2
krobison
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Have you looked into the Halo Genomics selector technology? This is probably around its sweet spot. If you wish to look at a very large number of individuals, Rain Dance might be a possibility as well.

Any of the capture oligo technologies (Illumina, Agilent, Nimblegen, etc) can operate in this range, of course. You haven't said how big "multiple" is, which could have a huge impact on picking a technology.
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Old 12-19-2011, 09:48 PM   #3
Marius
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I haven't loooked at these technologies yet..

Regarding "multiple": about 120 individuals, 100 kB each, with a good coverage if possible (50 x or so). Preferentially, I would have a single (ungapped) assembly of these 100bk for each indidivual specimen to then call SNPs..

Since I have easily access to an Illumina platform (HiSeq) and a Roche 454 platform (preferentially Illumina): is there a reliable to way to do this on these platforms?
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Old 12-20-2011, 05:48 AM   #4
Cofactor Genomics
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Hello,
Here is a paper that I was on which involved the capture of ~8kb fragments, however it could easily be adapted to capture 100kb. It takes a little more upfront work, but will provide many aliquots for capture.

http://www.ncbi.nlm.nih.gov/pubmed/21497292

Regards,

Jon
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Old 12-23-2011, 08:32 AM   #5
Marius
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Thank you!!! I actually found this kit by Illumina: TruSeq Custom Amplicon (TSCA).

Does anyone have experience with it? Is it well suited to e.g. sequence 4 different 96kb genomic fragments in multiple specimens? How is the quality? Coverage? - I'm happy for any experience and comments!
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