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

Emily Hodges, Michelle Rooks, Zhenyu Xuan, Arindam Bhattacharjee, D. Benjamin Gordon, Leonardo Brizuela, W. Richard McCombie, Gregory J. Hannon

Research output: Contribution to journalArticlepeer-review

133 Scopus citations

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 lg 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.

Original languageEnglish (US)
Pages (from-to)960-978
Number of pages19
JournalNature Protocols
Volume4
Issue number6
DOIs
StatePublished - 2009
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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