Article abstract
Nature Biotechnology 27, 182 - 189 (2009)
Published online: 1 February 2009 | doi:10.1038/nbt.1523
Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing
Andreas Gnirke1, Alexandre Melnikov1, Jared Maguire1, Peter Rogov1, Emily M LeProust2, William Brockman1,5, Timothy Fennell1, Georgia Giannoukos1, Sheila Fisher1, Carsten Russ1, Stacey Gabriel1, David B Jaffe1, Eric S Lander1,3,4 & Chad Nusbaum1
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.
- Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
- Agilent Technologies Inc., 5301 Stevens Creek Blvd., Santa Clara, California 95051, USA.
- Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.
- Department of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115, USA.
- Present address: Google, Inc., 5 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Correspondence to: Andreas Gnirke1 e-mail: gnirke@broad.mit.edu
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