Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency

• DOI: 10.1371/journal.pone.0143092
• Publication Date: 2015-11-25T13:47:48Z
• Authors: Luiz H. Araujo; Cynthia Timmers; Konstantin Shilo; Weiqiang Zhao; Jianying Zhang; Lianbo Yu; Thanemozhi G. Nat...; Clinton J. Miller; Ayse Selen Yilmaz; Tom Liu; Joseph Amann; José Roberto Lapa...; Carlos Gil Ferreira; David P. Carbone
• Keywords: GC-content

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated effect of pre-sequencing parameters to guide proper sample selection targeted gene Data from 113 FFPE lung tumor were collected, and sequencing was performed. Libraries constructed using custom probes paired-end sequenced on a next generation platform. A PCR-based quality control (QC) assay utilized determine quality, ratio generated in comparison DNA. We observed that storage time, PCR/QC ratio, input library preparation significantly correlated efficiency including depth coverage, alignment rate, insert size, read quality. combined score three proved highly accurate predict metrics. also showed wide count variability within genome, with worse coverage regions low GC content like KRAS. Sample had independent effects depth, worst results samples poor Our data confirm reliable cancer, provided adequate controls exercised. Tissue should be routinely assessed pre-analytical factors, may limited genomic if suboptimal utilized.