Abstract

The purpose of this study was to develop a massive parallel sequencing (MPS) workflow for diagnostic analysis of mismatch repair (MMR) genes using the GS Junior system (Roche). A pathogenic variant in one of four MMR genes, (MLH1, PMS2, MSH6, and MSH2), is the cause of Lynch Syndrome (LS), which mainly predispose to colorectal cancer. We used an amplicon-based sequencing method allowing specific and preferential amplification of the MMR genes including PMS2, of which several pseudogenes exist. The amplicons were pooled at different ratios to obtain coverage uniformity and maximize the throughput of a single-GS Junior run. In total, 60 previously identified and distinct variants (substitutions and indels), were sequenced by MPS and successfully detected. The heterozygote detection range was from 19% to 63% and dependent on sequence context and coverage. We were able to distinguish between false-positive and true-positive calls in homopolymeric regions by cross-sample comparison and evaluation of flow signal distributions. In addition, we filtered variants according to a predefined status, which facilitated variant annotation. Our study shows that implementation of MPS in routine diagnostics of LS can accelerate sample throughput and reduce costs without compromising sensitivity, compared to Sanger sequencing.

Highlights

  • Sanger sequencing (Sanger et al 1977) has been the gold standard for DNA sequencing during the past decades

  • We sequenced the four mismatch repair (MMR) genes MSH2, MLH1, MSH6, and PMS2 in 32 patients divided on four GS Junior runs

  • We evaluated if genetic testing using the GS Junior benchtop sequencer from Roche was suitable for implementation in a diagnostic laboratory in terms of sensitivity, specificity, distribution of coverage, hands-on time, costs, and sample turnaround time

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Summary

Introduction

Sanger sequencing (Sanger et al 1977) has been the gold standard for DNA sequencing during the past decades. MPS has especially been embraced by genomic research because it facilitates performance of complex genetic studies that were not technically or economically feasible with Sanger sequencing. In 2010, Roche introduced the GS Junior, which is a small benchtop sequencing platform more compatible with the needs of a diagnostic laboratory. This system produces 100,000 shotgun and 70,000 amplicon high-quality, filtered reads, each 10-h run. The performance of this system should be comparable to the well-documented GS FLX System a 2014 The Authors.

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