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

被引：1022

作者：

Gnirke, Andreas ^{[1
]}

Melnikov, Alexandre ^{[1
]}

Maguire, Jared ^{[1
]}

Rogov, Peter ^{[1
]}

LeProust, Emily M. ^{[2
]}

Brockman, William ^{[1
]}

Fennell, Timothy ^{[1
]}

Giannoukos, Georgia ^{[1
]}

Fisher, Sheila ^{[1
]}

Russ, Carsten ^{[1
]}

Gabriel, Stacey ^{[1
]}

Jaffe, David B. ^{[1
]}

Lander, Eric S. ^{[1
,3
,4
]}

Nusbaum, Chad ^{[1
]}

机构：

[1] Broad Inst MIT & Harvard, Cambridge, MA 02142 USA

[2] Agilent Technol, Santa Clara, CA 95051 USA

[3] MIT, Dept Biol, Cambridge, MA 02139 USA

[4] Harvard Univ, Sch Med, Dept Syst Biol, Boston, MA 02115 USA

来源：

NATURE BIOTECHNOLOGY | 2009年 / 27卷 / 02期

关键词：

MULTIPLEX AMPLIFICATION; GENOMIC SELECTION; DNA FRAGMENTS; LARGE SETS; PROBES; GENES;

D O I：

10.1038/nbt.1523

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

页码：182 / 189

页数：8

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