Department of Laboratory Medicine, Children and Women's Health, Faculty of Medicine, Norwegian University of Science and Technology Trondheim, Norway.
Department of Pathology and Medical Genetics, St. Olavs Hospital Trondheim, Norway.
Mol Genet Genomic Med. 2014 Mar;2(2):186-200. doi: 10.1002/mgg3.62. Epub 2014 Jan 21.
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.
本研究旨在开发一种使用 GS Junior 系统（罗氏）进行错配修复（MMR）基因诊断分析的大规模平行测序（MPS）工作流程。四个 MMR 基因（MLH1、PMS2、MSH6 和 MSH2）之一的致病性变异是林奇综合征（LS）的病因，LS 主要易患结直肠癌。我们使用基于扩增子的测序方法，允许对 MMR 基因（包括存在多个假基因的 PMS2）进行特异性和优先扩增。将扩增子以不同的比例混合，以获得覆盖均匀性并最大化单个 GS Junior 运行的通量。总共对 60 个先前鉴定的不同变异（取代和插入缺失）进行了 MPS 测序，并成功检测到。杂合子检测范围为 19%至 63%，取决于序列背景和覆盖度。我们能够通过交叉样本比较和评估流信号分布来区分同聚物区域中的假阳性和真阳性呼叫。此外，我们根据预定义的状态过滤变异，这有助于变异注释。我们的研究表明，与 Sanger 测序相比，在 LS 的常规诊断中实施 MPS 可以在不影响灵敏度的情况下加速样本通量并降低成本。
