Microdroplet-based PCR enrichment for large-scale targeted sequencing Export

@article{citeulike:6083224, abstract = {Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84\% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across 90\% of targeted bases; sequence variants were called with >99\% accuracy; and reproducibility between samples was high (r2 = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing.}, author = {Tewhey, Ryan and Warner, Jason B. and Nakano, Masakazu and Libby, Brian and Medkova, Martina and David, Patricia H. and Kotsopoulos, Steve K. and Samuels, Michael L. and Hutchison, J. Brian and Larson, Jonathan W. and Topol, Eric J. and Weiner, Michael P. and Harismendy, Olivier and Olson, Jeff and Link, Darren R. and Frazer, Kelly A.}, booktitle = {Nat Biotech}, citeulike-article-id = {6083224}, citeulike-linkout-0 = {http://dx.doi.org/10.1038/nbt.1583}, citeulike-linkout-1 = {http://dx.doi.org/10.1038/nbt.1583}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/19881494}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=19881494}, day = {01}, doi = {10.1038/nbt.1583}, issn = {1087-0156}, journal = {Nature Biotechnology}, keywords = {enrichment, microdroplet, pcr, resequencing, sequencing, targeted}, month = {November}, number = {11}, pages = {1025--1031}, posted-at = {2009-11-09 12:33:44}, priority = {2}, publisher = {Nature Publishing Group}, title = {Microdroplet-based PCR enrichment for large-scale targeted sequencing}, url = {http://dx.doi.org/10.1038/nbt.1583}, volume = {27}, year = {2009} }

TY - JOUR ID - citeulike:6083224 L3 - citeulike-article-id:6083224 N2 - Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across 90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r2 = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing. IS - 11 JF - Nature Biotechnology SN - 1087-0156 EP - 1031 TI - Microdroplet-based PCR enrichment for large-scale targeted sequencing PB - Nature Publishing Group VL - 27 T2 - Nat Biotech SP - 1025 KW - enrichment KW - microdroplet KW - pcr KW - resequencing KW - sequencing KW - targeted AU - Tewhey, Ryan AU - Warner, Jason AU - Nakano, Masakazu AU - Libby, Brian AU - Medkova, Martina AU - David, Patricia AU - Kotsopoulos, Steve AU - Samuels, Michael AU - Hutchison, Brian AU - Larson, Jonathan AU - Topol, Eric AU - Weiner, Michael AU - Harismendy, Olivier AU - Olson, Jeff AU - Link, Darren AU - Frazer, Kelly PY - 2009/11/01/ UR - http://dx.doi.org/10.1038/nbt.1583 ER -