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 journalArticle

128 Citations (Scopus)

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 - Jun 15 2009
Externally publishedYes

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High-Throughput Nucleotide Sequencing
Microarrays
Genes
Genome
Genomic Library
Gene Library
Base Pairing
Purification
Exons
Costs and Cost Analysis
DNA
Costs
Recognition (Psychology)

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Hodges, E., Rooks, M., Xuan, Z., Bhattacharjee, A., Gordon, D. B., Brizuela, L., ... Hannon, G. J. (2009). Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nature Protocols, 4(6), 960-978. https://doi.org/10.1038/nprot.2009.68

Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. / Hodges, Emily; Rooks, Michelle; Xuan, Zhenyu; Bhattacharjee, Arindam; Gordon, D. Benjamin; Brizuela, Leonardo; McCombie, W. Richard; Hannon, Gregory J.

In: Nature Protocols, Vol. 4, No. 6, 15.06.2009, p. 960-978.

Research output: Contribution to journalArticle

Hodges, E, Rooks, M, Xuan, Z, Bhattacharjee, A, Gordon, DB, Brizuela, L, McCombie, WR & Hannon, GJ 2009, 'Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing', Nature Protocols, vol. 4, no. 6, pp. 960-978. https://doi.org/10.1038/nprot.2009.68
Hodges, Emily ; Rooks, Michelle ; Xuan, Zhenyu ; Bhattacharjee, Arindam ; Gordon, D. Benjamin ; Brizuela, Leonardo ; McCombie, W. Richard ; Hannon, Gregory J. / Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. In: Nature Protocols. 2009 ; Vol. 4, No. 6. pp. 960-978.
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