title: Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform.
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 onAPC, BRAF, KRASandNRASgenes were validated by conventional sequencing.Results:In total, 34 kinds of mutations were detected including two novel mutations (FBXW7c.1740:C>G andSMAD4c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation (APC). The most frequently mutated genes wereAPC, TP53andKRASwith 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.
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary;; Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Bálint
authors.initials: B
authors.lastname: Péterfia
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kalmár
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Árpád V
authors.initials: ÁV
authors.lastname: Patai
authors.affiliation: Department of Physics of Complex Systems, Eötvös Loránd University, Budapest, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Csabai
authors.affiliation: Department of Physics of Complex Systems, Eötvös Loránd University, Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Bodor
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Micsik
authors.affiliation: Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Barnabás
authors.initials: B
authors.lastname: Wichmann
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Egedi
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary;; Tumor Progression Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Hollósi
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
authors.forename: Ilona
authors.initials: I
authors.lastname: Kovalszky
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary;; Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Tulassay
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary;; Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Béla
authors.initials: B
authors.lastname: Molnár
keywords: Targeted next generation sequencing - colorectal cancer - cancer genotyping - targeted cancer therapy - GS Junior - mutation hot-spots - mutation screening.