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BMC Genomics

, 15:184

Human and rodent genomics

Abstract

BackgroundNext 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.

ResultsWe 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.

ConclusionsHere 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.

KeywordsNext generation sequencing Target enrichment Sequence capture Quantitative PCR NCI60 Mutation detection AbbreviationsqPCRQuantitative polymerase chain reaction

CqQuantitation cycle

SNPSingle nucleotide polymorphism.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-15-184 contains supplementary material, which is available to authorized users.

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Autor: Bram De Wilde - Steve Lefever - Wes Dong - Jude Dunne - Syed Husain - Stefaan Derveaux - Jan Hellemans - Jo Vandesompele

Fuente: https://link.springer.com/







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