Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing
- 3 November 2011
- journal article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 6 (12), 1870-1886
- https://doi.org/10.1038/nprot.2011.396
Abstract
The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussedKeywords
This publication has 26 references indexed in Scilit:
- Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinomaNature, 2011
- Frequent Mutation of BAP1 in Metastasizing Uveal MelanomasScience, 2010
- A de novo paradigm for mental retardationNature Genetics, 2010
- A map of human genome variation from population-scale sequencingNature, 2010
- Mutation discovery by targeted genomic enrichment of multiplexed barcoded samplesNature Methods, 2010
- Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variantsNature Genetics, 2010
- Target-enrichment strategies for next-generation sequencingNature Methods, 2010
- Sequencing technologies — the next generationNature Reviews Genetics, 2009
- Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencingNature Protocols, 2009
- The diploid genome sequence of an Asian individualNature, 2008