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 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.

Gene promoter and exon DNA methylation changes in colon cancer development – mRNA expression and tumor mutation alterations

“…The mutation frequencies of a panel consisting 12 CRC-associated genes in CRC and AD samples were measured in our previous multiplex PCR-based CRC mutation hot-spot sequencing study [ 27 ]. DNA methylation alterations were also detected in the mutation hot-spot regions of 12 analyzed CRC-associated genes that are frequently mutated, including TP53 , APC , KRAS , BRAF and FBXW7 .…”

“…Methylation percentage values are shown in 100 base pair analysis regions located in mutation hot-spot areas of genes ( TP53, APC, KRAS, BRAF and FBXW7 ) frequently mutated in CRC and adenoma tissue. The frequencies of mutations in CRC and adenoma samples detected in our previous multiplex PCR-based CRC mutation hot-spot sequencing study [ 27 ] are also represented. * p < 0.05, CRC = colorectal cancer, Ad = adenoma, N = normal adjacent tissue

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“…Using normal, benign and malignant colorectal tissue samples, mutation hot-spot regions of 12 CRC-associated genes ( APC , B-Raf proto-oncogene, serine/threonine kinase ( BRAF ), catenin beta 1 ( CTNNB1 ), epidermal growth factor receptor ( EGFR ), F-box and WD repeat domain containing 7 ( FBXW7 ), KRAS proto-oncogene, GTPase ( KRAS ), NRAS proto-oncogene, GTPase ( NRAS ), mutS homolog 6 ( MSH6 ), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ), SMAD family member 2 and 4 ( SMAD2 and SMAD4 ), tumor protein 53 ( TP53 )) were amplified using a custom-made multiplex PCR panel previously designed by our research group [ 27 ]. Amplicon sequencing was carried out on a GS Junior instrument (Roche) using ligated and barcoded adaptors as described earlier [ 27 ]. Bead enrichment and sequencing were performed using GS Junior Titanium Sequencing Kit (Roche) according to the Sequencing Method Manual, GS FLX Titanium Series.…”

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples

“…However, selectively sequencing pathogens of interest provides better sensitivity, better specificity, ease of downstream analysis, and lower cost by allowing more samples to be tested in one run ( 10 ). Targeted NGS has been applied successfully in cancer diagnostics ( 11 – 13 ). All of these advantages suggest that targeted NGS can be used for syndromic testing by providing a comprehensive diagnostic assay for the detection of known, clinically relevant pathogens from a variety of specimens, particularly for cases that present nonspecific disease signs that may be associated with multiple infectious agents.…”

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

“…The Acknowledgments section in our paper 1 was incomplete and should instead read as follows: The results published here are in whole or part based upon data generated by The Cancer Genome Atlas managed by the NCI and NHGRI ( http://cancergenome.nih.gov ). The TCGA data analyzed here are available through dbGAP, accession phs000178.v9.p8 ( http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000178.v9.p8 ).…”