Journal
NATURE BIOTECHNOLOGY
Volume 27, Issue 2, Pages 182-189Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.1523
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Funding
- National Human Genome Research Institute [HG03067-05]
- funds of the Broad Institute
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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 similar to 60% of target bases in the exonic 'catch', and similar to 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.
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