Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing

被引：133

作者：

Hodges, Emily ^{[1
,2
]}

Rooks, Michelle ^{[1
,2
]}

Xuan, Zhenyu ^{[1
]}

Bhattacharjee, Arindam ^{[3
]}

Gordon, D. Benjamin ^{[3
]}

Brizuela, Leonardo ^{[3
]}

McCombie, W. Richard ^{[1
]}

Hannon, Gregory J. ^{[1
,2
]}

机构：

[1] Cold Spring Harbor Lab, Watson Sch Biol Sci, Cold Spring Harbor, NY 11724 USA

[2] Cold Spring Harbor Lab, Howard Hughes Med Inst, Cold Spring Harbor, NY 11724 USA

[3] Agilent Technol, Santa Clara, CA USA

来源：

NATURE PROTOCOLS | 2009年 / 4卷 / 06期

关键词：

CAPTURE; GENES; SETS;

D O I：

10.1038/nprot.2009.68

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Complementary techniques that deepen information content and minimize reagent costs are required to realize the full potential of massively parallel sequencing. Here, we describe a resequencing approach that directs focus to genomic regions of high interest by combining hybridization-based purification of multi-megabase regions with sequencing on the Illumina Genome Analyzer (GA). The capture matrix is created by a microarray on which probes can be programmed as desired to target any non-repeat portion of the genome, while the method requires only a basic familiarity with microarray hybridization. We present a detailed protocol suitable for 1-2 mu g of input genomic DNA and highlight key design tips in which high specificity (>65% of reads stem from enriched exons) and high sensitivity (98% targeted base pair coverage) can be achieved. We have successfully applied this to the enrichment of coding regions, in both human and mouse, ranging from 0.5 to 4 Mb in length. From genomic DNA library production to base-called sequences, this procedure takes approximately 9-10 d inclusive of array captures and one Illumina flow cell run.

引用

页码：960 / 974

页数：15

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