Homo sapiens Genome sequencing

The utilization of archived, formalin-fixed paraffin embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here we perform whole genome sequencing of DNA isolated from 2 triple-negative breast cancer tumors archived for greater than 11 years as 5 uCm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C>T or G>A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.

由于DNA损伤及正常间质污染，利用存档的福尔马林固定石蜡包埋（formalin-fixed paraffin embedded, FFPE）肿瘤样本开展大规模并行测序一直颇具挑战。本研究对2例存档时长超过11年的5 uCm FFPE切片三阴性乳腺癌肿瘤样本的分离DNA，以及匹配的种系DNA开展全基因组测序。与种系样本相比，肿瘤样本的FFPE损伤DNA测序读段（reads）数量存在差异，其比对错配率相对较高，且富集C>T或G>A碱基替换。仅需低至100万条读段即可检测到错配率的升高，这使得我们能够在全基因组测序前提前评估样本完整性。通过应用整合了全局核苷酸错配率与局部错配率的创新性质量过滤策略，我们开发出一种即使在FFPE诱导的DNA损伤存在下，仍可识别高置信度体细胞突变的方法。该方法得到的乳腺癌突变谱与既往研究结果一致，且揭示了潜在具有重要功能意义的突变。本研究证实，对样本量有限的FFPE存档肿瘤（如治疗后残留癌或转移性活检组织）开展全基因组深度测序分析具有可行性。