AI Article Synopsis
- A new resequencing method combines hybridization-based purification with Illumina sequencing to focus on important genomic regions, reducing costs and increasing information content.
- The process involves creating a customized capture matrix using a microarray to target specific non-repeat areas of the genome, and it’s user-friendly with basic microarray knowledge.
- The protocol efficiently enriches coding regions in both human and mouse DNA, achieving over 65% specificity and 98% sensitivity within a timeframe of about 9-10 days.
Article 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 microg 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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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990409 | PMC |
| http://dx.doi.org/10.1038/nprot.2009.68 | DOI Listing |
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