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

Gnirke et al. present a bead-based method for capturing sequences of interest in the human genome for massively parallel sequencing. Using long, biotinylated RNA probes to pull down PCR-amplified DNA fragments, they demonstrate sequencing of 2.5 Mbs of exons in 1,900 genes. 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.