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BMC Genomics. 2014 Mar 10;15:184. doi: 10.1186/1471-2164-15-184.

Target enrichment using parallel nanoliter quantitative PCR amplification.

Author information

  • 1Center of Medical Genetics Ghent, Ghent University, Ghent, Belgium. Joke.Vandesompele@ugent.be.

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.

PMID:
24612714
PMCID:
PMC4234423
DOI:
10.1186/1471-2164-15-184
[PubMed - indexed for MEDLINE]
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