Target enrichment using parallel nanoliter quantitative PCR amplification

BMC Genomics

Jo Vandesompele⁴  Jan Hellemans⁴  Stefaan Derveaux¹  Syed Husain³  Jude Dunne³  Wes Dong³  Steve Lefever²  Bram De Wilde² 

[1]WaferGen Biosystems Europe S.à r.l, Luxembourg, Luxembourg

[2]Center of Medical Genetics Ghent, Ghent University, Ghent, Belgium

[3]WaferGen Biosystems Inc, Fremont, USA

[4]Biogazelle, Zwijnaarde, Belgium

关键词: Mutation detection;    NCI60;    Quantitative PCR;    Sequence capture;    Target enrichment;    Next generation sequencing;   

Others  :  1217808
DOI  :  10.1186/1471-2164-15-184

 received in 2013-07-30, accepted in 2014-02-25,  发布年份 2014

【 摘 要 】

Background

Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high.

Results

We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform.

Conclusions

Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform’s promise as a targeted resequencing method for multi-gene routine sequencing diagnostics.

【 授权许可】

   
2014 De Wilde et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Mardis ER: The impact of next-generation sequencing technology on genetics. Trends Genet 2008, 24:133-141.
• [2]Mardis E, Ding L, Dooling D, Larson D, McLellan M, Chen K, Koboldt D, Fulton R, Delehaunty K, McGrath S, Fulton L, Locke D, Magrini V, Abbott R, Vickery T, Reed J, Robinson J, Wylie T, Smith S, Carmichael L, Eldred J, Harris C, Walker J, Peck J, Du F, Dukes A, Sanderson G, Brummett A, Clark E, McMichael J, et al.: Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009, 361:1058-1066.
• [3]Pleasance ED, Keira Cheetham R, Stephens PJ, Mcbride DJ, Humphray SJ, Greenman CD, Varela I, Lin M-L, Ordóñez GR, Bignell GR, Ye K, Alipaz J, Bauer MJ, Beare D, Butler A, Carter RJ, Chen L, Cox AJ, Edkins S, Kokko-Gonzales PI, Gormley NA, Grocock RJ, Haudenschild CD, Hims MM, James T, Jia M, Kingsbury Z, Leroy C, Marshall J, Menzies A, et al.: A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 2010, 463:191-196.
• [4]Pleasance ED, Stephens PJ, O'meara S, Mcbride DJ, Meynert A, Jones D, Lin M-L, Beare D, Lau KW, Greenman C, Varela I, Nik-Zainal S, Davies HR, Ordoñez GR, Mudie LJ, Latimer C, Edkins S, Stebbings L, Chen L, Jia M, Leroy C, Marshall J, Menzies A, Butler A, Teague JW, Mangion J, Sun YA, McLaughlin SF, Peckham HE, Tsung EF, et al.: A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature 2010, 463:184-190.
• [5]Bell D, Berchuck A, Birrer M, Chien J, Cramer DW, Dao F, Dhir R, DiSaia P, Gabra H, Glenn P, Godwin AK, Gross J, Hartmann L, Huang M, Huntsman DG, Iacocca M, Imielinski M, Kalloger S, Karlan BY, Levine DA, Mills GB, Morrison C, Mutch D, Olvera N, Orsulic S, Park K, Petrelli N, Rabeno B, Rader JS, Sikic BI, et al.: Integrated genomic analyses of ovarian carcinoma. Nature 2011, 474:609-615.
• [6]Larsen LA, Christiansen M, Vuust J, Andersen PS: High throughput mutation screening by automated capillary electrophoresis. Comb Chem High Throughput Screen 2000, 3:393-409.
• [7]Mitchelson KR: The use of capillary electrophoresis for DNA polymorphism analysis. Mol Biotechnol 2003, 24:41-68.
• [8]Shoemaker RH: The NCI60 human tumour cell line anticancer drug screen. Nat Genet 2006, 6:813-823.
• [9]Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, Jia M, Shepherd R, Leung K, Menzies A, Teague JW, Campbell PJ, Stratton MR, Futreal PA: COSMIC: mining complete cancer genomes in the catalogue of somatic mutations in cancer. Nucleic Acids Res 2010, 39:D945-D950. Database
• [10]Li H, Durbin R: Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 2009, 25:1754-1760.
• [11]McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010, 20:1297-1303.
• [12]Ikediobi ON, Davies H, Bignell G, Edkins S, Stevens C, O'Meara S, Santarius T, Avis T, Barthorpe S, Brackenbury L, Buck G, Butler A, Clements J, Cole J, Dicks E, Forbes S, Gray K, Halliday K, Harrison R, Hills K, Hinton J, Hunter C, Jenkinson A, Jones D, Kosmidou V, Lugg R, Menzies A, Mironenko T, Parker A, Perry J, et al.: Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther 2006, 5:2606-2612.
• [13]Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, Beroukhim R, Milner DA, Granter SR, Du J, Lee C, Wagner SN, Li C, Golub TR, Rimm DL, Meyerson ML, Fisher DE, Sellers WR: Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005, 436:117-122.
• [14]Abaan OD, Polley EC, Davis SR, Zhu YJ, Bilke S, Walker RL, Pineda M, Gindin Y, Jiang Y, Reinhold WC, Holbeck SL, Simon RM, Doroshow JH, Pommier Y, Meltzer PS: The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology. Cancer Res 2013, 73:4372-4382.
• [15]Mertes F, Elsharawy A, Sauer S, van Helvoort JMLM, van der Zaag PJ, Franke A, Nilsson M, Lehrach H, Brookes AJ: Targeted enrichment of genomic DNA regions for next-generation sequencing. Bfgoxfordjournalsorg 2011, 10:374-386.
• [16]Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ: Target-enrichment strategies for next-generation sequencing. Nat Meth 2010, 7:111-118.
• [17]Dahl A, Sultan M, Jung A, Schwartz R, Lange M, Steinwand M, Livak KJ, Lehrach H, Nyarsik L: Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips. Biomed Microdevices 2007, 9:307-314.
• [18]Mertes F, Biens K, Lehrach H, Wagner M, Dahl A: High-throughput universal probe salmonella serotyping (UPSS) by nanoPCR. J Microbiol Methods 2010, 83:217-223.
• [19]Jones MA, Bhide S, Chin E, Ng BG, Rhodenizer D, Zhang VW, Sun JJ, Tanner A, Freeze HH, Hegde MR: Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation. Genet Med 2011, 13:921-932.
• [20]Halbritter J, Diaz K, Chaki M, Porath JD, Tarrier B, Fu C, Innis JL, Allen SJ, Lyons RH, Stefanidis CJ, Omran H, Soliman NA, Otto EA: High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing. J Med Genet 2012, 49:756-767.
• [21]Hollants S, Redeker EJW, Matthijs G: Microfluidic amplification as a tool for massive parallel sequencing of the familial hypercholesterolemia genes. Clin Chem 2012, 58:717-724.
• [22]Schlipf NA, Schüle R, Klimpe S, Karle KN, Synofzik M, Schicks J, Riess O, Schöls L, Bauer P: Amplicon-based high-throughput pooled sequencing identifies mutations in CYP7B1 and SPG7 in sporadic spastic paraplegia patients. Clin Genet 2011, 80:148-160.
• [23]Hu H, Wrogemann K, Kalscheuer V, Tzschach A, Richard H, Haas SA, Menzel C, Bienek M, Froyen G, Raynaud M, Van Bokhoven H, Chelly J, Ropers H, Chen W: Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing. HUGO J 2010, 3:41-49.
• [24]Mondal K, Shetty AC, Patel V, Cutler DJ, Zwick ME: Targeted sequencing of the human X chromosome exome. Genomics 2011, 98:260-265.
• [25]Schrauwen I, Sommen M, Corneveaux JJ, Reiman RA, Hackett NJ, Claes C, Claes K, Bitner-Glindzicz M, Coucke P, Van Camp G, Huentelman MJ: A sensitive and specific diagnostic test for hearing loss using a microdroplet PCR-based approach and next generation sequencing. Am J Med Genet 2012, 161:145-152.
• [26]Valencia CA, Ankala A, Rhodenizer D, Bhide S, Littlejohn MR, Keong LM, Rutkowski A, Sparks S, Bonnemann C, Hegde M: Comprehensive mutation analysis for congenital muscular dystrophy: a clinical PCR-based enrichment and next-generation sequencing panel. PLoS ONE 2013, 8:e53083.
• [27]Johansson H, Isaksson M, Sorqvist EF, Roos F, Stenberg J, Sjoblom T, Botling J, Micke P, Edlund K, Fredriksson S, Kultima HG, Ericsson O, Nilsson M: Targeted resequencing of candidate genes using selector probes. Nucleic Acids Res 2011, 39:e8-e8.
• [28]Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JKV, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE: The consensus coding sequences of human breast and colorectal cancers. Science 2006, 314:268-274.