Office of Research and Development, U.S. Environmental Protection Agency, MD-B105-03, 109 T.W. Alexander Drive, Research Triangle Park, NC, USA.
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA.
Sci Rep. 2022 Apr 20;12(1):6509. doi: 10.1038/s41598-022-10301-0.
Formalin fixation of biological specimens damages nucleic acids and limits their use in genomic analyses. Previously, we showed that RNA isolation with an organocatalyst (2-amino-5-methylphenyl phosphonic acid, used to speed up reversal of formalin-induced adducts) and extended heated incubation (ORGΔ) improved RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples. The primary goal of this study was to evaluate whether ORGΔ treatment improves DNA-sequencing data from clinical FFPE samples. We isolated RNA and DNA ± ORGΔ from paired FFPE and frozen human renal and ovarian carcinoma specimens collected as part of the National Cancer Institute Biospecimen Pre-analytical Variables program. Tumor types were microscopically confirmed from adjacent tissue sections. Following extraction, DNA was fragmented and sequenced and differences were compared between frozen and FFPE sample pairs. Treatment with ORGΔ improved concurrent SNP calls in FFPE DNA compared to non-ORGΔ FFPE samples and enhanced confidence in SNP calls for all FFPE DNA samples, beyond that of matched frozen samples. In general, the concordant SNPs identified in paired frozen and FFPE DNA samples agreed for both genotype and homozygosity vs. heterozygosity of calls regardless of ORGΔ treatment. The increased confidence in ORGΔ FFPE DNA variant calls relative to the matched frozen DNA suggests a novel application of this method. With further optimization, this method may improve quality of DNA-sequencing data in FFPE as well as frozen tissue samples.
福尔马林固定的生物标本会损害核酸，并限制其在基因组分析中的应用。先前，我们表明使用有机催化剂（2-氨基-5-甲基苯膦酸，用于加速逆转福尔马林诱导的加合物）和延长加热孵育（ORGΔ）可以改善福尔马林固定石蜡包埋（FFPE）组织样本的 RNA 测序数据。本研究的主要目标是评估 ORGΔ 处理是否能改善临床 FFPE 样本的 DNA 测序数据。我们从国家癌症研究所生物样本预分析变量计划中收集的配对 FFPE 和冷冻人肾和卵巢癌标本中分离 RNA 和 DNA±ORGΔ。从相邻组织切片中显微镜确认肿瘤类型。提取后，对 DNA 进行片段化和测序，并比较冷冻和 FFPE 样本对之间的差异。与非 ORGΔ FFPE 样本相比，ORGΔ 处理可改善 FFPE 中 DNA 的 SNP 调用，并增强所有 FFPE DNA 样本的 SNP 调用的置信度，超出了匹配的冷冻样本的置信度。一般来说，无论 ORGΔ 处理如何，在配对的冷冻和 FFPE DNA 样本中识别出的一致 SNP 都与基因型和纯合子与杂合子的调用一致。与匹配的冷冻 DNA 相比，ORGΔ FFPE DNA 变体调用的置信度增加表明了该方法的新应用。通过进一步优化，该方法可能会提高 FFPE 以及冷冻组织样本中 DNA 测序数据的质量。
