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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.!
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.!
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