Salipante Stephen J, Scroggins Sheena M, Hampel Heather L, Turner Emily H, Pritchard Colin C
Department of Laboratory Medicine, University of Washington, Seattle WA;
Department of Internal Medicine, Division of Human Genetics, The Ohio State University, Columbus, OH.
Clin Chem. 2014 Sep;60(9):1192-9. doi: 10.1373/clinchem.2014.223677. Epub 2014 Jun 30.
Microsatellite instability (MSI) is a useful phenotype in cancer diagnosis and prognosis. Nevertheless, methods to detect MSI status from next generation DNA sequencing (NGS) data are underdeveloped.
We developed an approach to detect the MSI phenotype using NGS (mSINGS). The method was used to evaluate mononucleotide microsatellite loci that were incidentally sequenced after targeted gene enrichment and could be applied to gene or exome capture panels designed for other purposes. For each microsatellite locus, the number of differently sized repeats in experimental samples were quantified and compared to a population of normal controls. Loci were considered unstable if the experimental number of repeats was statistically greater than in the control population. MSI status was determined by the fraction of unstable microsatellite loci.
We examined data from 324 samples generated using targeted gene capture assays of 3 different sizes, ranging from a 0.85-Mb to a 44-Mb exome design and incorporating from 15 to 2957 microsatellite markers. When we compared mSING results to MSI-PCR as a gold standard for 108 cases, we found the approach to be both diagnostically sensitive (range of 96.4% to 100% across 3 panels) and specific (range of 97.2% to 100%) for determining MSI status. The fraction of unstable microsatellite markers calculated from sequencing data correlated with the number of unstable loci detected by conventional MSI-PCR testing.
NGS data can enable highly accurate detection of MSI, even from limited capture designs. This novel approach offers several advantages over existing PCR-based methods.
微卫星不稳定性（MSI）在癌症诊断和预后评估中是一种有用的表型。然而，从下一代DNA测序（NGS）数据中检测MSI状态的方法仍不完善。
我们开发了一种利用NGS检测MSI表型的方法（mSINGS）。该方法用于评估在靶向基因富集后偶然测序的单核苷酸微卫星位点，并且可应用于为其他目的设计的基因或外显子捕获面板。对于每个微卫星位点，对实验样品中不同大小重复序列的数量进行定量，并与正常对照群体进行比较。如果实验中的重复序列数量在统计学上大于对照群体，则该位点被认为是不稳定的。MSI状态由不稳定微卫星位点的比例确定。
我们检查了来自324个样本的数据，这些样本使用了3种不同大小的靶向基因捕获检测方法生成，外显子设计范围从0.85 Mb到44 Mb，并包含15至2957个微卫星标记。当我们将mSINGS结果与作为108例病例金标准的MSI-PCR进行比较时，我们发现该方法在确定MSI状态方面具有诊断敏感性（3个面板的范围为96.4%至100%）和特异性（范围为97.2%至100%）。从测序数据计算出的不稳定微卫星标记比例与通过传统MSI-PCR检测检测到的不稳定位点数量相关。
即使来自有限的捕获设计，NGS数据也能够实现对MSI的高精度检测。这种新方法相对于现有的基于PCR的方法具有几个优点。
