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Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies

Sachin Sah, Liangjing Chen, Jeffrey Houghton, Jon Kemppainen, Adam C Marko, Robert Zeigler and Gary J Latham*

Author Affiliations

Asuragen, Inc, Austin, TX 78744, USA

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Genome Medicine 2013, 5:77  doi:10.1186/gm481

Published: 30 August 2013

Abstract

The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay (‘QFI-PCR’) that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing.