Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

TY - JOUR

T1 - Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

AU - Harakalova, M.

AU - Mokry, M.

AU - Hrdlickova, B.

AU - Renkens, I.

AU - Duran, K.J.

AU - van Roekel, H.

AU - Lansu, N.

AU - van Roosmalen, M.

AU - de Bruijn, E.

AU - Nijman, I.J.

AU - Kloosterman, W.P.

AU - Cuppen, E.

N1 - Reporting year: 2011

PY - 2011

Y1 - 2011

N2 - 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 discussed.

AB - 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 discussed.

U2 - 10.1038/nprot.2011.396

DO - 10.1038/nprot.2011.396

M3 - Article

SN - 1754-2189

VL - 6

SP - 1870

EP - 1886

JO - Nature Protocols

JF - Nature Protocols

IS - 12

ER -