Protocol abstract
Nature Protocols 4, - 960 - 974 (2009)
Published online: 28 May 2009 | doi:10.1038/nprot.2009.68
Subject Categories: Genetic analysis | Genomics and proteomics | Nucleic acid based molecular biology
Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing
Emily Hodges1,2, Michelle Rooks1,2, Zhenyu Xuan1, Arindam Bhattacharjee3, D Benjamin Gordon3, Leonardo Brizuela3, W Richard McCombie1 & Gregory J Hannon1,2
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.
- Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
- Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
- Agilent Technologies Inc., Santa Clara, California, USA.
Correspondence to: Gregory J Hannon1,2 e-mail: hannon@cshl.edu
nature-products
A-Z product listing
- 0.2 ml 8-well PCR strip tubes(VWR)
- 1.5 ml centrifuge tubes(Eppendorf)
- 1.7 ml maximum recovery centrifuge tubes(Axygen)
- 2
Phusion High-Fidelity PCR Master Mix(Finnzymes) - 30-G syringes(BD Medical)
- 50 bp DNA Ladder(Crystalgen)
