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

Hodges, E., Rooks, M., Xuan, Z., Bhattacharjee, A., Gordon, D. B., Brizuela, L., McCombie, W. R., Hannon, G. J. (2009) Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nature Protocols, 4 (6). pp. 960-974. ISSN 1754-2189

URL: http://www.ncbi.nlm.nih.gov/pubmed/19478811
DOI: 10.1038/nprot.2009.68

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 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.

Item Type: Paper
Uncontrolled Keywords: Genetic analysis Genomics and proteomics Nucleic acid based molecular biology
Subjects: bioinformatics > genomics and proteomics > databases > database construction
bioinformatics > genomics and proteomics > Validation and Standardization > Microarray Data Standardization and Validation
bioinformatics > genomics and proteomics > analysis and processing > microarray gene expression processing
bioinformatics > genomics and proteomics > annotation > sequence annotation
bioinformatics > genomics and proteomics > analysis and processing > Sequence Data Processing
bioinformatics > genomics and proteomics > Mapping and Rendering > Sequence Rendering
Investigative techniques and equipment > probes
CSHL Authors:
Communities: CSHL labs > Hannon lab
CSHL labs > McCombie lab
Depositing User: Editor Margaret Fantz
Date Deposited: 17 Apr 2012 18:52
Last Modified: 11 Apr 2013 19:18
PMCID: PMC2990409
Related URLs:
URI: http://repository.cshl.edu/id/eprint/26108

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