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Open Access Highly Accessed Methodology article

Target enrichment using parallel nanoliter quantitative PCR amplification

Bram De Wilde1, Steve Lefever1, Wes Dong2, Jude Dunne2, Syed Husain2, Stefaan Derveaux3, Jan Hellemans4 and Jo Vandesompele14*

Author Affiliations

1 Center of Medical Genetics Ghent, Ghent University, Ghent, Belgium

2 WaferGen Biosystems Inc, Fremont, USA

3 WaferGen Biosystems Europe S.à r.l, Luxembourg, Luxembourg

4 Biogazelle, Zwijnaarde, Belgium

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BMC Genomics 2014, 15:184  doi:10.1186/1471-2164-15-184

Published: 10 March 2014

Abstract

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.

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