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  • Solution hybrid selection w...
    Jaffe, David B; Giannoukos, Georgia; Melnikov, Alexandre; Maguire, Jared; Fisher, Sheila; Fennell, Timothy; Gnirke, Andreas; Lander, Eric S; Brockman, William; Nusbaum, Chad; Rogov, Peter; Russ, Carsten; LeProust, Emily M; Gabriel, Stacey

    Nature biotechnology, 02/2009, Letnik: 27, Številka: 2
    Journal Article

    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.