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

来自 Semantic Scholar

阅读量：

49

作者：

E Hodges，M Rooks，Z Xuan，A Bhattacharjee，D Benjamin Gordon，L Brizuela，W Richard Mccombie，GJ Hannon

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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 microg 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.

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关键词：

RNA polymerase Regulon Sigma factor Microarray

DOI：

10.1038/nprot.2009.68

被引量：

493

年份：

2009

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