title: Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing.
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 discussed.
authors: Department of Medical Genetics, University Medical Center Utrecht, The Netherlands.
authors: Magdalena
authors: M
authors: Harakalova
authors: Michal
authors: M
authors: Mokry
authors: Barbara
authors: B
authors: Hrdlickova
authors: Ivo
authors: I
authors: Renkens
authors: Karen
authors: K
authors: Duran
authors: Henk
authors: H
authors: van Roekel
authors: Nico
authors: N
authors: Lansu
authors: Mark
authors: M
authors: van Roosmalen
authors: Ewart
authors: E
authors: de Bruijn
authors: Isaac J
authors: IJ
authors: Nijman
authors: Wigard P
authors: WP
authors: Kloosterman
authors: Edwin
authors: E
authors: Cuppen
mesh_terms: D004247:DNA / Q000737:chemistry*; D058893:DNA Barcoding, Taxonomic; D015723:Gene Library; D016678:Genome; D023281:Genomics; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA