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Nucleic Acids Research 2005 33(18):e152; doi:10.1093/nar/gni152
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Published online 12 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Shotgun haplotyping: a novel method for surveying allelic sequence variation

Sarah J. Lindsay, James K. Bonfield and Matthew E. Hurles*

Wellcome Trust Sanger Institute Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

*To whom correspondence should be addressed. Tel: +44 (0) 1223 495377; Fax +44 (0) 1223 494919; Email: meh{at}sanger.ac.uk

Received June 24, 2005. Revised September 19, 2005. Accepted September 19, 2005.

Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.

DDBJ/EMBL/GenBank accession numbers: DQ075317, DQ075318, DQ075319, DQ075320


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