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Nat Biotechnol. 2009 Feb;27(2):182-9. doi: 10.1038/nbt.1523. Epub 2009 Feb 1.

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

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1
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. gnirke@broad.mit.edu

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 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.

PMID:
19182786
PMCID:
PMC2663421
DOI:
10.1038/nbt.1523
[Indexed for MEDLINE]
Free PMC Article
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