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


ABSTRACT: 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.

SUBMITTER: Hodges E 

PROVIDER: S-EPMC2990409 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Hodges Emily E   Rooks Michelle M   Xuan Zhenyu Z   Bhattacharjee Arindam A   Benjamin Gordon D D   Brizuela Leonardo L   Richard McCombie W W   Hannon Gregory J GJ  

Nature protocols 20090528 6


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  ...[more]

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