Microdroplet-based PCR enrichment for large-scale targeted sequencing

被引：332

作者：

Tewhey, Ryan ^{[1
,2
]}

Warner, Jason B. ^{[3
]}

Nakano, Masakazu ^{[1
]}

Libby, Brian ^{[3
]}

Medkova, Martina ^{[3
]}

David, Patricia H. ^{[3
]}

Kotsopoulos, Steve K. ^{[3
]}

Samuels, Michael L. ^{[3
]}

Hutchison, J. Brian ^{[3
]}

Larson, Jonathan W. ^{[3
]}

Topol, Eric J. ^{[1
]}

Weiner, Michael P. ^{[3
]}

Harismendy, Olivier ^{[1
]}

Olson, Jeff ^{[3
]}

Link, Darren R. ^{[3
]}

Frazer, Kelly A. ^{[1
]}

机构：

[1] Scripps Res Inst, Scripps Translat Sci Inst, La Jolla, CA 92037 USA

[2] Univ Calif San Diego, Div Biol Sci, La Jolla, CA 92093 USA

[3] RainDance Technol, Lexington, MA USA

来源：

NATURE BIOTECHNOLOGY | 2009年 / 27卷 / 11期

关键词：

HUMAN GENOME; MULTIPLEX AMPLIFICATION; EXON CAPTURE; SELECTION;

D O I：

10.1038/nbt.1583

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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 similar to 90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r(2) = 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.

引用

页码：1025 / U94

页数：10

共 26 条

[1] Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels [J].

Ahn, Keunho ;

Agresti, Jeremy ;

Chong, Henry ;

Marquez, Manuel ;

Weitz, D. A. .

APPLIED PHYSICS LETTERS, 2006, 88 (26)

[2] Direct selection of human genomic loci by microarray hybridization [J].

Albert, Thomas J. ;

Molla, Michael N. ;

Muzny, Donna M. ;

Nazareth, Lynne ;

Wheeler, David ;

Song, Xingzhi ;

Richmond, Todd A. ;

Middle, Chris M. ;

Rodesch, Matthew J. ;

Packard, Charles J. ;

Weinstock, George M. ;

Gibbs, Richard A. .

NATURE METHODS, 2007, 4 (11) :903-905

[3] A haplotype map of the human genome [J].

Altshuler, D ;

Brooks, LD ;

Chakravarti, A ;

Collins, FS ;

Daly, MJ ;

Donnelly, P ;

Gibbs, RA ;

Belmont, JW ;

Boudreau, A ;

Leal, SM ;

Hardenbol, P ;

Pasternak, S ;

Wheeler, DA ;

Willis, TD ;

Yu, FL ;

Yang, HM ;

Zeng, CQ ;

Gao, Y ;

Hu, HR ;

Hu, WT ;

Li, CH ;

Lin, W ;

Liu, SQ ;

Pan, H ;

Tang, XL ;

Wang, J ;

Wang, W ;

Yu, J ;

Zhang, B ;

Zhang, QR ;

Zhao, HB ;

Zhao, H ;

Zhou, J ;

Gabriel, SB ;

Barry, R ;

Blumenstiel, B ;

Camargo, A ;

Defelice, M ;

Faggart, M ;

Goyette, M ;

Gupta, S ;

Moore, J ;

Nguyen, H ;

Onofrio, RC ;

Parkin, M ;

Roy, J ;

Stahl, E ;

Winchester, E ;

Ziaugra, L ;

Shen, Y .

NATURE, 2005, 437 (7063) :1299-1320

[4] Formation of dispersions using "flow focusing" in microchannels [J].

Anna, SL ;

Bontoux, N ;

Stone, HA .

APPLIED PHYSICS LETTERS, 2003, 82 (03) :364-366

[5] Accurate whole human genome sequencing using reversible terminator chemistry [J].

Bentley, David R. ;

Balasubramanian, Shankar ;

Swerdlow, Harold P. ;

Smith, Geoffrey P. ;

Milton, John ;

Brown, Clive G. ;

Hall, Kevin P. ;

Evers, Dirk J. ;

Barnes, Colin L. ;

Bignell, Helen R. ;

Boutell, Jonathan M. ;

Bryant, Jason ;

Carter, Richard J. ;

Cheetham, R. Keira ;

Cox, Anthony J. ;

Ellis, Darren J. ;

Flatbush, Michael R. ;

Gormley, Niall A. ;

Humphray, Sean J. ;

Irving, Leslie J. ;

Karbelashvili, Mirian S. ;

Kirk, Scott M. ;

Li, Heng ;

Liu, Xiaohai ;

Maisinger, Klaus S. ;

Murray, Lisa J. ;

Obradovic, Bojan ;

Ost, Tobias ;

Parkinson, Michael L. ;

Pratt, Mark R. ;

Rasolonjatovo, Isabelle M. J. ;

Reed, Mark T. ;

Rigatti, Roberto ;

Rodighiero, Chiara ;

Ross, Mark T. ;

Sabot, Andrea ;

Sankar, Subramanian V. ;

Scally, Aylwyn ;

Schroth, Gary P. ;

Smith, Mark E. ;

Smith, Vincent P. ;

Spiridou, Anastassia ;

Torrance, Peta E. ;

Tzonev, Svilen S. ;

Vermaas, Eric H. ;

Walter, Klaudia ;

Wu, Xiaolin ;

Zhang, Lu ;

Alam, Mohammed D. ;

Anastasi, Carole .

NATURE, 2008, 456 (7218) :53-59

[6] Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project [J].

Birney, Ewan ;

Stamatoyannopoulos, John A. ;

Dutta, Anindya ;

Guigo, Roderic ;

Gingeras, Thomas R. ;

Margulies, Elliott H. ;

Weng, Zhiping ;

Snyder, Michael ;

Dermitzakis, Emmanouil T. ;

Stamatoyannopoulos, John A. ;

Thurman, Robert E. ;

Kuehn, Michael S. ;

Taylor, Christopher M. ;

Neph, Shane ;

Koch, Christoph M. ;

Asthana, Saurabh ;

Malhotra, Ankit ;

Adzhubei, Ivan ;

Greenbaum, Jason A. ;

Andrews, Robert M. ;

Flicek, Paul ;

Boyle, Patrick J. ;

Cao, Hua ;

Carter, Nigel P. ;

Clelland, Gayle K. ;

Davis, Sean ;

Day, Nathan ;

Dhami, Pawandeep ;

Dillon, Shane C. ;

Dorschner, Michael O. ;

Fiegler, Heike ;

Giresi, Paul G. ;

Goldy, Jeff ;

Hawrylycz, Michael ;

Haydock, Andrew ;

Humbert, Richard ;

James, Keith D. ;

Johnson, Brett E. ;

Johnson, Ericka M. ;

Frum, Tristan T. ;

Rosenzweig, Elizabeth R. ;

Karnani, Neerja ;

Lee, Kirsten ;

Lefebvre, Gregory C. ;

Navas, Patrick A. ;

Neri, Fidencio ;

Parker, Stephen C. J. ;

Sabo, Peter J. ;

Sandstrom, Richard ;

Shafer, Anthony .

NATURE, 2007, 447 (7146) :799-816

[7] Comprehensive genomic characterization defines human glioblastoma genes and core pathways [J].

Chin, L. ;

Meyerson, M. ;

Aldape, K. ;

Bigner, D. ;

Mikkelsen, T. ;

VandenBerg, S. ;

Kahn, A. ;

Penny, R. ;

Ferguson, M. L. ;

Gerhard, D. S. ;

Getz, G. ;

Brennan, C. ;

Taylor, B. S. ;

Winckler, W. ;

Park, P. ;

Ladanyi, M. ;

Hoadley, K. A. ;

Verhaak, R. G. W. ;

Hayes, D. N. ;

Spellman, Paul T. ;

Absher, D. ;

Weir, B. A. ;

Ding, L. ;

Wheeler, D. ;

Lawrence, M. S. ;

Cibulskis, K. ;

Mardis, E. ;

Zhang, Jinghui ;

Wilson, R. K. ;

Donehower, L. ;

Wheeler, D. A. ;

Purdom, E. ;

Wallis, J. ;

Laird, P. W. ;

Herman, J. G. ;

Schuebel, K. E. ;

Weisenberger, D. J. ;

Baylin, S. B. ;

Schultz, N. ;

Yao, Jun ;

Wiedemeyer, R. ;

Weinstein, J. ;

Sander, C. ;

Gibbs, R. A. ;

Gray, J. ;

Kucherlapati, R. ;

Lander, E. S. ;

Myers, R. M. ;

Perou, C. M. ;

McLendon, Roger .

NATURE, 2008, 455 (7216) :1061-1068

[8] Somatic mutations affect key pathways in lung adenocarcinoma [J].

Ding, Li ;

Getz, Gad ;

Wheeler, David A. ;

Mardis, Elaine R. ;

McLellan, Michael D. ;

Cibulskis, Kristian ;

Sougnez, Carrie ;

Greulich, Heidi ;

Muzny, Donna M. ;

Morgan, Margaret B. ;

Fulton, Lucinda ;

Fulton, Robert S. ;

Zhang, Qunyuan ;

Wendl, Michael C. ;

Lawrence, Michael S. ;

Larson, David E. ;

Chen, Ken ;

Dooling, David J. ;

Sabo, Aniko ;

Hawes, Alicia C. ;

Shen, Hua ;

Jhangiani, Shalini N. ;

Lewis, Lora R. ;

Hall, Otis ;

Zhu, Yiming ;

Mathew, Tittu ;

Ren, Yanru ;

Yao, Jiqiang ;

Scherer, Steven E. ;

Clerc, Kerstin ;

Metcalf, Ginger A. ;

Ng, Brian ;

Milosavljevic, Aleksandar ;

Gonzalez-Garay, Manuel L. ;

Osborne, John R. ;

Meyer, Rick ;

Shi, Xiaoqi ;

Tang, Yuzhu ;

Koboldt, Daniel C. ;

Lin, Ling ;

Abbott, Rachel ;

Miner, Tracie L. ;

Pohl, Craig ;

Fewell, Ginger ;

Haipek, Carrie ;

Schmidt, Heather ;

Dunford-Shore, Brian H. ;

Kraja, Aldi ;

Crosby, Seth D. ;

Sawyer, Christopher S. .

NATURE, 2008, 455 (7216) :1069-1075

[9] Human genetic variation and its contribution to complex traits [J].

Frazer, Kelly A. ;

Murray, Sarah S. ;

Schork, Nicholas J. ;

Topol, Eric J. .

NATURE REVIEWS GENETICS, 2009, 10 (04) :241-251

[10] Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing [J].

Gnirke, Andreas ;

Melnikov, Alexandre ;

Maguire, Jared ;

Rogov, Peter ;

LeProust, Emily M. ;

Brockman, William ;

Fennell, Timothy ;

Giannoukos, Georgia ;

Fisher, Sheila ;

Russ, Carsten ;

Gabriel, Stacey ;

Jaffe, David B. ;

Lander, Eric S. ;

Nusbaum, Chad .

NATURE BIOTECHNOLOGY, 2009, 27 (02) :182-189

← 1 2 3 →