High-throughput droplet digital PCR system for absolute quantitation of DNA copy number

Benjamin J Hindson, Kevin D Ness, Donald A Masquelier, Phillip Belgrader, Nicholas J Heredia, Anthony J Makarewicz, Isaac J Bright, Michael Y Lucero, Amy L Hiddessen, Tina C Legler, Tyler K Kitano, Michael R Hodel, Jonathan F Petersen, Paul W Wyatt, Erin R Steenblock, Pallavi H Shah, Luc J Bousse, Camille B Troup, Jeffrey C Mellen, Dean K Wittmann, Nicholas G Erndt, Thomas H Cauley, Ryan T Koehler, Austin P So, Simant Dube, Klint A Rose, Luz Montesclaros, Shenglong Wang, David P Stumbo, Shawn P Hodges, Steven Romine, Fred P Milanovich, Helen E White, John F Regan, George A Karlin-Neumann, Christopher M Hindson, Serge Saxonov, Bill W Colston, Benjamin J Hindson, Kevin D Ness, Donald A Masquelier, Phillip Belgrader, Nicholas J Heredia, Anthony J Makarewicz, Isaac J Bright, Michael Y Lucero, Amy L Hiddessen, Tina C Legler, Tyler K Kitano, Michael R Hodel, Jonathan F Petersen, Paul W Wyatt, Erin R Steenblock, Pallavi H Shah, Luc J Bousse, Camille B Troup, Jeffrey C Mellen, Dean K Wittmann, Nicholas G Erndt, Thomas H Cauley, Ryan T Koehler, Austin P So, Simant Dube, Klint A Rose, Luz Montesclaros, Shenglong Wang, David P Stumbo, Shawn P Hodges, Steven Romine, Fred P Milanovich, Helen E White, John F Regan, George A Karlin-Neumann, Christopher M Hindson, Serge Saxonov, Bill W Colston

Abstract

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Figures

Figure 1
Figure 1
Droplet digital PCR workflow: (a) Samples and droplet generation oil are loaded into an eight-channel droplet generator cartridge. (b) A vacuum is applied to the droplet well, which draws sample and oil through a flow-focusing nozzle where monodisperse 1 nL droplets are formed. In

Figure 2

Determination of copy number variation…

Figure 2

Determination of copy number variation states by droplet digital PCR. (a) Measured copy…

Figure 2
Determination of copy number variation states by droplet digital PCR. (a) Measured copy number variation states in HapMap samples for MRGPRX1, Chromosome X, CYP2D6, and (b) CCL3L1. (c) Correlation of measured copy number alterations of GRB7 and ERBB2 in DNA extracted from normal and tumorous breast tissues. Each marker represents a CNV measurement from a single ddPCR well of ∼20 000 droplets. Error bars indicate the Poisson 95% confidence intervals for each copy number determination.

Figure 3

Detection of the BRAF V600E…

Figure 3

Detection of the BRAF V600E rare mutant allele in the presence of homologous…

Figure 3
Detection of the BRAF V600E rare mutant allele in the presence of homologous wildtype DNA by droplet digital PCR. Serial dilutions of the mutant cell line DNA were prepared in a constant background of wildtype human genomic DNA. Droplet partitioning reduces competitive amplification effects allowing detection down to 0.001% mutant fraction, 1 000 times lower than real-time PCR. The mutant cell line contains 35% BRAF V600E, as measured by ddPCR.

Figure 4

Absolute quantitation of circulating fetal…

Figure 4

Absolute quantitation of circulating fetal and maternal DNA from cell-free plasma for male…

Figure 4
Absolute quantitation of circulating fetal and maternal DNA from cell-free plasma for male and female fetuses. (a) Quantitation of fetal DNA concentration using SRY (red bar) and hypermethylated RASSF1 (blue bar). The RASSF1 gene of circulating fetal DNA is hypermethylated whereas maternal DNA is hypomethylated. Methylation sensitive restriction enzymes selectively digested away the hypomethylated fraction, leaving the hypermethylated fetal DNA that was quantified. (b) Quantitation of total DNA concentration (black bar) represented as the weighted average from six independent assay measurements including undigested RASSF1 and β-actin as well as RNaseP and TERT. (c) Fetal loads as determined from the ratio of SRY to total (male fetuses only) and RASSF1 to total (male and female fetuses). For male fetuses, the Pearson’s correlation coefficient between SRY and RASSF1 fetal loads was 97.3%. Fetal DNA is not completely hypermethylated; therefore, the RASSF1 fetal loads measured for some samples are lower than those determined using SRY. Error bars represent the Poisson 95% confidence intervals of the concentration or the ratio in the case of fetal load estimates.
Figure 2
Figure 2
Determination of copy number variation states by droplet digital PCR. (a) Measured copy number variation states in HapMap samples for MRGPRX1, Chromosome X, CYP2D6, and (b) CCL3L1. (c) Correlation of measured copy number alterations of GRB7 and ERBB2 in DNA extracted from normal and tumorous breast tissues. Each marker represents a CNV measurement from a single ddPCR well of ∼20 000 droplets. Error bars indicate the Poisson 95% confidence intervals for each copy number determination.
Figure 3
Figure 3
Detection of the BRAF V600E rare mutant allele in the presence of homologous wildtype DNA by droplet digital PCR. Serial dilutions of the mutant cell line DNA were prepared in a constant background of wildtype human genomic DNA. Droplet partitioning reduces competitive amplification effects allowing detection down to 0.001% mutant fraction, 1 000 times lower than real-time PCR. The mutant cell line contains 35% BRAF V600E, as measured by ddPCR.
Figure 4
Figure 4
Absolute quantitation of circulating fetal and maternal DNA from cell-free plasma for male and female fetuses. (a) Quantitation of fetal DNA concentration using SRY (red bar) and hypermethylated RASSF1 (blue bar). The RASSF1 gene of circulating fetal DNA is hypermethylated whereas maternal DNA is hypomethylated. Methylation sensitive restriction enzymes selectively digested away the hypomethylated fraction, leaving the hypermethylated fetal DNA that was quantified. (b) Quantitation of total DNA concentration (black bar) represented as the weighted average from six independent assay measurements including undigested RASSF1 and β-actin as well as RNaseP and TERT. (c) Fetal loads as determined from the ratio of SRY to total (male fetuses only) and RASSF1 to total (male and female fetuses). For male fetuses, the Pearson’s correlation coefficient between SRY and RASSF1 fetal loads was 97.3%. Fetal DNA is not completely hypermethylated; therefore, the RASSF1 fetal loads measured for some samples are lower than those determined using SRY. Error bars represent the Poisson 95% confidence intervals of the concentration or the ratio in the case of fetal load estimates.

References

    1. Sykes P. J.; Neoh S. H.; Brisco M. J.; Hughes E.; Condon J.; Morley A. A. Biotechniques 1992, 13, 444–449.
    1. Vogelstein B.; Kinzler K. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9236–9241.
    1. McCaughan F.; Dear P. H. J. Pathol. 2010, 220, 297–306.
    1. Dube S.; Qin J.; Ramakrishnan R. PLoS One 2008, 3, e2876.
    1. Morrison T.; Hurley J.; Garcia J.; Yoder K.; Katz A.; Roberts D.; Cho J.; Kanigan T.; Ilyin S. E.; Horowitz D.; Dixon J. M.; Brenan C. J. Nucleic Acids Res. 2006, 34, e123.
    1. Warren L.; Bryder D.; Weissman I. L.; Quake S. R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17807–17812.
    1. Ottesen E. A.; Hong J. W.; Quake S. R.; Leadbetter J. R. Science 2006, 314, 1464–1467.
    1. Fan H. C.; Quake S. R. Anal. Chem. 2007, 79, 7576–7579.
    1. Dressman D.; Yan H.; Traverso G.; Kinzler K. W.; Vogelstein B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8817–8822.
    1. Diehl F.; Li M.; He Y.; Kinzler K. W.; Vogelstein B.; Dressman D. Nat. Methods 2006, 3, 551–559.
    1. Diehl F.; Diaz L. A. Jr. Curr. Opin. Oncol. 2007, 19, 36–42.
    1. Li M.; Diehl F.; Dressman D.; Vogelstein B.; Kinzler K. W. Nat. Methods 2006, 3, 95–97.
    1. Li M.; Chen W. D.; Papadopoulos N.; Goodman S. N.; Bjerregaard N. C.; Laurberg S.; Levin B.; Juhl H.; Arber N.; Moinova H.; Durkee K.; Schmidt K.; He Y.; Diehl F.; Velculescu V. E.; Zhou S.; Diaz L. A. Jr.; Kinzler K. W.; Markowitz S. D.; Vogelstein B.; Nat. Biotechnol. 2009, 27(9), 858−863.
    1. Beer N. R.; Hindson B. J.; Wheeler E. K.; Hall S. B.; Rose K. A.; Kennedy I. M.; Colston B. W. Anal. Chem. 2007, 79, 8471–8475.
    1. Beer N. R.; Wheeler E. K.; Lee-Houghton L.; Watkins N.; Nasarabadi S.; Hebert N.; Leung P.; Arnold D. W.; Bailey C. G.; Colston B. W. Anal. Chem. 2008, 80, 1854–1858.
    1. Kiss M. M.; Ortoleva-Donnelly L.; Beer N. R.; Warner J.; Bailey C. G.; Colston B. W.; Rothberg J. M.; Link D. R.; Leamon J. H. Anal. Chem. 2008, 80, 8975–8981.
    1. Weaver S.; Dube S.; Mir A.; Qin J.; Sun G.; Ramakrishnan R.; Jones R. C.; Livak K. J. Methods 2010, 50, 271–276.
    1. Gonzalez E.; Kulkarni H.; Bolivar H.; Mangano A.; Sanchez R.; Catano G.; Nibbs R. J.; Freedman B. I.; Quinones M. P.; Bamshad M. J.; Murthy K. K.; Rovin B. H.; Bradley W.; Clark R. A.; Anderson S. A.; O’Connell R J.; Agan B. K.; Ahuja S. S.; Bologna R.; Sen L.; Dolan M. J.; Ahuja S. K. Science 2005, 307, 1434–1440.
    1. Sudmant P. H.; Kitzman J. O.; Antonacci F.; Alkan C.; Malig M.; Tsalenko A.; Sampas N.; Bruhn L.; Shendure J.; Eichler E. E. Science 2010, 330, 641–646.
    1. Carter N. P. Nat. Genet. 2007, 39, S16–21.
    1. Schouten J. P.; McElgunn C. J.; Waaijer R.; Zwijnenburg D.; Diepvens F.; Pals G. Nucleic Acids Res. 2002, 30, e57.
    1. Aldhous M. C.; Abu Bakar S.; Prescott N. J.; Palla R.; Soo K.; Mansfield J. C.; Mathew C. G.; Satsangi J.; Armour J. A. Hum. Mol. Genet. 2010, 19, 4930–4938.
    1. Sherry S. T.; Ward M. H.; Kholodov M.; Baker J.; Phan L.; Smigielski E. M.; Sirotkin K. Nucleic Acids Res. 2001, 29, 308–311.
    1. Qin J.; Jones R. C.; Ramakrishnan R. Nucleic Acids Res. 2008, 36, e116.
    1. Alkan C.; Kidd J. M.; Marques-Bonet T.; Aksay G.; Antonacci F.; Hormozdiari F.; Kitzman J. O.; Baker C.; Malig M.; Mutlu O.; Sahinalp S. C.; Gibbs R. A.; Eichler E. E. Nat. Genet. 2009, 41, 1061–1067.
    1. Bartlett J. M.; Ibrahim M.; Jasani B.; Morgan J. M.; Ellis I.; Kay E.; Connolly Y.; Campbell F.; O’Grady A.; Barnett S.; Miller K. Amer. J. Clin. Pathol. 2009, 131, 106–111.
    1. Kauraniemi P.; Kuukasjarvi T.; Sauter G.; Kallioniemi A. Am. J. Pathol. 2003, 163, 1979–1984.
    1. Luoh S. W. Cancer Genet. Cytogenet. 2002, 136, 43–47.
    1. Benlloch S.; Paya A.; Alenda C.; Bessa X.; Andreu M.; Jover R.; Castells A.; Llor X.; Aranda F. I.; Massuti B. J. Mol. Diagn. 2006, 8, 540–543.
    1. Lo Y. M.; Corbetta N.; Chamberlain P. F.; Rai V.; Sargent I. L.; Redman C. W.; Wainscoat J. S. Lancet 1997, 350, 485–487.
    1. Wright C. F.; Burton H. Hum. Reprod. Update 2009, 15, 139–151.
    1. Pathak A. K.; Bhutani M.; Kumar S.; Mohan A.; Guleria R. Clin. Chem. 2006, 52, 1833–1842.
    1. Fan H. C.; Blumenfeld Y. J.; Chitkara U.; Hudgins L.; Quake S. R. Clin. Chem. 2010, 56, 1279–1286.
    1. Tong Y. K.; Jin S.; Chiu R. W.; Ding C.; Chan K. C.; Leung T. Y.; Yu L.; Lau T. K.; Lo Y. M. Clin. Chem. 2010, 56, 90–98.
    1. Fan H. C.; Blumenfeld Y. J.; Chitkara U.; Hudgins L.; Quake S. R. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16266–16271.
    1. Hosono N.; Kubo M.; Tsuchiya Y.; Sato H.; Kitamoto T.; Saito S.; Ohnishi Y.; Nakamura Y. Hum. Mutat. 2008, 29, 182–189.
    1. Fan H. C.; Blumenfeld Y. J.; El-Sayed Y. Y.; Chueh J.; Quake S. R. Am. J. Obstet. Gynecol. 2009, 200 (543), e541–547.

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner