Moores UCSD Cancer Center, Bioinformatics and Systems Biology Graduate Program, Department of Pediatrics and Rady Children's Hospital, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Nucleic Acids Res. 2012 Aug;40(14):e107. doi: 10.1093/nar/gks299. Epub 2012 Apr 6.
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 two triple-negative breast cancer tumors archived for >11 years as 5 µm 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 · G > T · 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 损伤和正常基质的污染，利用存档的福尔马林固定石蜡包埋（FFPE）肿瘤样本进行大规模平行测序一直具有挑战性。在这里，我们对从两个存档超过 11 年的三阴性乳腺癌肿瘤中分离的 DNA 进行全基因组测序，这些肿瘤作为 5 µm FFPE 切片和匹配的种系 DNA 进行了测序。肿瘤样本显示出不同数量的 FFPE 损伤 DNA 测序reads，与种系样本相比，相对较高的比对错配率富集了 C·G>T·A 取代。这种错配率的增加在仅使用 100 万reads 时即可观察到，从而可以在全基因组测序之前对样本完整性进行预先评估。通过应用创新的质量过滤，包括全局核苷酸错配率和局部错配率，我们提出了一种方法，即使在存在 FFPE 诱导的 DNA 损伤的情况下，也可以识别高可信度的体细胞突变。这导致了与先前研究一致的乳腺癌突变特征，并揭示了潜在的重要功能突变。我们的研究表明，对治疗后残留的癌症或转移性活检等有限样本量的 FFPE 存档肿瘤进行全基因组深度测序分析是可行的。
