Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours

Scientific Reports
Stephen Q WongAlexander Dobrovic

Abstract

Massively parallel sequencing offers the ability to interrogate a tumour biopsy for multiple mutational changes. For clinical samples, methodologies must enable maximal extraction of available sequence information from formalin-fixed and paraffin-embedded (FFPE) material. We assessed the use of targeted capture for mutation detection in FFPE DNA. The capture probes targeted the coding region of all known kinase genes and selected oncogenes and tumour suppressor genes. Seven melanoma cell lines and matching FFPE xenograft DNAs were sequenced. An informatics pipeline was developed to identify variants and contaminating mouse reads. Concordance of 100% was observed between unfixed and formalin-fixed for reported COSMIC variants including BRAF V600E. mutations in genes not conventionally screened including ERBB4, ATM, STK11 and CDKN2A were readily detected. All regions were adequately covered with independent reads regardless of GC content. This study indicates that hybridisation capture is a robust approach for massively parallel sequencing of FFPE samples.

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Citations

Sep 12, 2015·The Journal of Molecular Diagnostics : JMD·Lotte N J MoensJohan Botling
Sep 6, 2015·The Journal of Pediatrics·Hoon-Chul KangJoseph G Gleeson
Aug 19, 2014·Cell Proliferation·D SekarS Saravanan
Nov 26, 2014·Clinical Chemistry·Hongdo Do, Alexander Dobrovic
Jul 23, 2020·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·William Mathieson, Geraldine A Thomas
Jan 1, 2016·Cancers of the Head & Neck·Tim N Beck, Erica A Golemis
Jul 25, 2017·Genome Biology·Gahee ParkDonghyun Park
Mar 2, 2017·International Journal of Genomics·Pan ZhangYan Guo
Mar 6, 2021·Nature Communications·Ismael A VergaraMark Shackleton
Jul 30, 2021·BMC Medical Genomics·Seung-Ho ShinDonghyun Park

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Methods Mentioned

BETA
exome sequencing
biopsies
xenograft
xenografts
HT144
PCR
deamination
amplicon-based sequencing
scraping

Software Mentioned

Illumina
GATK
BWA
BLAT
Genome Analysis Tool Kit ( GATK )
CASAVA
COSMIC
GATK Unified

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