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

A Gnirke, A Melnikov, J Maguire, P Rogov… - Nature …, 2009 - nature.com
A Gnirke, A Melnikov, J Maguire, P Rogov, EM LeProust, W Brockman, T Fennell…
Nature biotechnology, 2009nature.com
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 …
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.
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