Journal
NATURE PROTOCOLS
Volume 4, Issue 6, Pages 960-974Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.68
Keywords
-
Categories
Funding
- NIH
- Stanley Foundation
- Howard Hughes Medical Institute
- NATIONAL CANCER INSTITUTE [P01CA013106] Funding Source: NIH RePORTER
Ask authors/readers for more resources
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 mu 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available