Hodges, E., Rooks, M., Xuan, Z., Bhattacharjee, A., Gordon, D. B., Brizuela, L., McCombie, W. R., Hannon, G. J. (May 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
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.
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