Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform

Bálint Péterfia; Alexandra Kalmár; Árpád V Patai; István Csabai; András Bodor; Tamás Micsik; Barnabás Wichmann; Krisztina Egedi; Péter Hollósi; Ilona Kovalszky; Zsolt Tulassay; Béla Molnár

doi:10.7150/jca.16037

Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform

J Cancer. 2017 Jan 12;8(2):162-173. doi: 10.7150/jca.16037. eCollection 2017.

Authors

Affiliations

¹ 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary;; Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
² 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
³ Department of Physics of Complex Systems, Eötvös Loránd University, Budapest, Hungary.
⁴ 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
⁵ Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
⁶ 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary;; Tumor Progression Research Group, Hungarian Academy of Sciences, Budapest, Hungary.

Abstract

Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations (FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation (APC). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument.

Keywords: Targeted next generation sequencing - colorectal cancer - cancer genotyping - targeted cancer therapy - GS Junior - mutation hot-spots - mutation screening..