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- Title
Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.
- Authors
Gnirke, Andreas; Melnikov, Alexandre; Maguire, Jared; Rogov, Peter; LeProust, Emily M; Brockman, William; Fennell, Timothy; Giannoukos, Georgia; Fisher, Sheila; Russ, Carsten; Gabriel, Stacey; Jaffe, David B; Lander, Eric S; Nusbaum, Chad
- Abstract
Targeting 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 ∼60% of target bases in the exonic 'catch', and ∼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.
- Subjects
HUMAN genome; NUCLEOTIDE sequence; OLIGONUCLEOTIDES; DNA; EXONS (Genetics); BIOTECHNOLOGY; BIOTECHNOLOGY experiments; BIOTECHNOLOGY research
- Publication
Nature Biotechnology, 2009, Vol 27, Issue 2, p182
- ISSN
1087-0156
- Publication type
Article
- DOI
10.1038/nbt.1523