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Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.

Publication TypeJournal Article
AuthorsGnirke, A., Melnikov A., Maguire J., Rogov P., LeProust EM, Brockman W., Fennell T., Giannoukos G., Fisher S., Russ C., Gabriel S., Jaffe DB, Lander E. S., and Nusbaum C.
AbstractTargeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.
Year of Publication2009
JournalNature biotechnology
Volume27
Issue2
Pages182-9
Date Published (YYYY/MM/DD)2009/02/01
ISSN Number1087-0156
DOI10.1038/nbt.1523
PubMedhttp://www.ncbi.nlm.nih.gov/pubmed/19182786?dopt=Abstract