Microfluidic droplet enrichment for targeted sequencing

被引：40

作者：

Eastburn, Dennis J. ^{[1
,2
]}

Huang, Yong ^{[3
,4
]}

Pellegrino, Maurizio ^{[2
]}

Sciambi, Adam ^{[1
,2
]}

Ptacek, Louis J. ^{[3
,4
]}

Abate, Adam R. ^{[1
]}

机构：

[1] Univ Calif San Francisco, Calif Inst Quantitat Biosci, Dept Bioengn & Therapeut Sci, San Francisco, CA 94158 USA

[2] Mission Bio Inc, San Francisco, CA 94107 USA

[3] Univ Calif San Francisco, Dept Neurol, San Francisco, CA 94158 USA

[4] Univ Calif San Francisco, Howard Hughes Med Inst, San Francisco, CA 94158 USA

来源：

NUCLEIC ACIDS RESEARCH | 2015年 / 43卷 / 13期

基金：

美国国家科学基金会; 美国国家卫生研究院;

关键词：

VARIANT DETECTION; HUMAN GENOME; ACCURATE; DNA; SELECTION; PCR; GENERATION; MUTATIONS; LIBRARIES; CAPTURE;

D O I：

10.1093/nar/gkv297

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.

引用

页数：8

共 43 条

[1] Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition [J].

Adey, Andrew ;

Morrison, Hilary G. ;

Asan ;

Xun, Xu ;

Kitzman, Jacob O. ;

Turner, Emily H. ;

Stackhouse, Bethany ;

MacKenzie, Alexandra P. ;

Caruccio, Nicholas C. ;

Zhang, Xiuqing ;

Shendure, Jay .

GENOME BIOLOGY, 2010, 11 (12)

[2] Accurate and comprehensive sequencing of personal genomes [J].

Ajay, Subramanian S. ;

Parker, Stephen C. J. ;

Abaan, Hatice Ozel ;

Fajardo, Karin V. Fuentes ;

Margulies, Elliott H. .

GENOME RESEARCH, 2011, 21 (09) :1498-1505

[3] 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

[4] An integrated map of genetic variation from 1,092 human genomes [J].

Altshuler, David M. ;

Durbin, Richard M. ;

Abecasis, Goncalo R. ;

Bentley, David R. ;

Chakravarti, Aravinda ;

Clark, Andrew G. ;

Donnelly, Peter ;

Eichler, Evan E. ;

Flicek, Paul ;

Gabriel, Stacey B. ;

Gibbs, Richard A. ;

Green, Eric D. ;

Hurles, Matthew E. ;

Knoppers, Bartha M. ;

Korbel, Jan O. ;

Lander, Eric S. ;

Lee, Charles ;

Lehrach, Hans ;

Mardis, Elaine R. ;

Marth, Gabor T. ;

McVean, Gil A. ;

Nickerson, Deborah A. ;

Schmidt, Jeanette P. ;

Sherry, Stephen T. ;

Wang, Jun ;

Wilson, Richard K. ;

Gibbs, Richard A. ;

Dinh, Huyen ;

Kovar, Christie ;

Lee, Sandra ;

Lewis, Lora ;

Muzny, Donna ;

Reid, Jeff ;

Wang, Min ;

Wang, Jun ;

Fang, Xiaodong ;

Guo, Xiaosen ;

Jian, Min ;

Jiang, Hui ;

Jin, Xin ;

Li, Guoqing ;

Li, Jingxiang ;

Li, Yingrui ;

Li, Zhuo ;

Liu, Xiao ;

Lu, Yao ;

Ma, Xuedi ;

Su, Zhe ;

Tai, Shuaishuai ;

Tang, Meifang .

NATURE, 2012, 491 (7422) :56-65

[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] Genomics: catch me if you can [J].

Blow, Nathan .

NATURE METHODS, 2009, 6 (07) :539-542

[7]

Bodi K, 2013, J Biomol Tech, V24, P73, DOI 10.7171/jbt.13-2402-002

[8] Performance comparison of exome DNA sequencing technologies [J].

Clark, Michael J. ;

Chen, Rui ;

Lam, Hugo Y. K. ;

Karczewski, Konrad J. ;

Chen, Rong ;

Euskirchen, Ghia ;

Butte, Atul J. ;

Snyder, Michael .

NATURE BIOTECHNOLOGY, 2011, 29 (10) :908-U206

[9] Detailed molecular characterisation of acute myeloid leukaemia with a normal karyotype using targeted DNA capture [J].

Conte, N. ;

Varela, I. ;

Grove, C. ;

Manes, N. ;

Yusa, K. ;

Moreno, T. ;

Segonds-Pichon, A. ;

Bench, A. ;

Gudgin, E. ;

Herman, B. ;

Bolli, N. ;

Ellis, P. ;

Haddad, D. ;

Costeas, P. ;

Rad, R. ;

Scott, M. ;

Huntly, B. ;

Bradley, A. ;

Vassiliou, G. S. .

LEUKEMIA, 2013, 27 (09) :1820-1825

[10] Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping [J].

de Vree, Paula J. P. ;

de Wit, Elzo ;

Yilmaz, Mehmet ;

de Heijning, Monique van ;

Klous, Petra ;

Verstegen, Marjon J. A. M. ;

Wan, Yi ;

Teunissen, Hans ;

Krijger, Peter H. L. ;

Geeven, Geert ;

Eijk, Paul P. ;

Sie, Daoud ;

Ylstra, Bauke ;

Hulsman, Lorette O. M. ;

van Dooren, Marieke F. ;

van Zutven, Laura J. C. M. ;

van den Ouweland, Ans ;

Verbeek, Sjef ;

van Dijk, Ko Willems ;

Cornelissen, Marion ;

Das, Atze T. ;

Ben Berkhout ;

Sikkema-Raddatz, Birgit ;

van den Berg, Eva ;

van der Vlies, Pieter ;

Weening, Desiree ;

den Dunnen, Johan T. ;

Matusiak, Magdalena ;

Lamkanfi, Mohamed ;

Ligtenberg, Marjolijn J. L. ;

ter Brugge, Petra ;

Jonkers, Jos ;

Foekens, John A. ;

Martens, John W. ;

van der Luijt, Rob ;

van Amstel, Hans Kristian Ploos ;

van Min, Max ;

Splinter, Erik ;

de Laat, Wouter .

NATURE BIOTECHNOLOGY, 2014, 32 (10) :1019-+

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