Target enrichment using parallel nanoliter quantitative PCR amplification

被引：16

作者：

De Wilde, Bram ^{[1
]}

Lefever, Steve ^{[1
]}

Dong, Wes ^{[2
]}

Dunne, Jude ^{[2
]}

Husain, Syed ^{[2
]}

Derveaux, Stefaan ^{[3
]}

Hellemans, Jan ^{[4
]}

Vandesompele, Jo ^{[1
,4
]}

机构：

[1] Univ Ghent, Ctr Med Genet Ghent, B-9000 Ghent, Belgium

[2] WaferGen Biosyst Inc, Fremont, CA USA

[3] WaferGen Biosyst Europe Sarl, Luxembourg, Luxembourg

[4] Biogazelle, Zwijnaarde, Belgium

来源：

BMC GENOMICS | 2014年 / 15卷

关键词：

Next generation sequencing; Target enrichment; Sequence capture; Quantitative PCR; NCI60; Mutation detection; CAPILLARY-ELECTROPHORESIS; MUTATION ANALYSIS; GENOMIC ANALYSES; CANCER GENOME; GENERATION; GENES;

D O I：

10.1186/1471-2164-15-184

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：

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.

引用

页数：14

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