Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL), Dover Air Force Base, DE, United States; ARP Sciences, LLC, Rockville, MD, United States.
Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL), Dover Air Force Base, DE, United States; ARP Sciences, LLC, Rockville, MD, United States.
Forensic Sci Int Genet. 2018 May;34:257-264. doi: 10.1016/j.fsigen.2018.02.015. Epub 2018 Feb 19.
DNA sequence damage from cytosine deamination is well documented in degraded samples, such as those from ancient and forensic contexts. This study examined the effect of a DNA repair treatment on mitochondrial DNA (mtDNA) from aged and degraded skeletal samples. DNA extracts from 21 non-probative, degraded skeletal samples (aged 50-70 years) were utilized for the analysis. A portion of each sample extract was subjected to DNA repair using a commercial repair kit, the New England BioLabs' NEBNext FFPE DNA Repair Kit (Ipswich, MA). MtDNA was enriched using PCR and targeted capture in a side-by-side experiment of untreated and repaired DNA. Sequencing was performed using both traditional (Sanger-type; STS) and next-generation sequencing (NGS) methods Although cytosine deamination was evident in the mtDNA sequence data, the observed level of damaged bases varied by sequencing method as well as by enrichment type. The STS PCR amplicon data did not show evidence of cytosine deamination that could be distinguished from background signal in either the untreated or repaired sample set. However, the same PCR amplicons showed 850 C → T/G → A substitutions consistent with cytosine deamination with variant frequencies (VFs) of up to 25% when sequenced using NGS methods The occurrence of base misincorporation due to cytosine deamination was reduced by 98% (to 10) in the NGS amplicon data after repair. The NGS capture data indicated low levels (1-2%) of cytosine deamination in mtDNA fragments that was effectively mitigated by DNA repair. The observed difference in the level of cytosine deamination between the PCR and capture enrichment methods can be attributed to the greater propensity for stochastic effects from the PCR enrichment technique employed (e.g., low template input, increased PCR cycles). Altogether these results indicate that DNA repair may be required when sequencing PCR-amplified DNA from degraded forensic case samples with NGS methods.
DNA 序列损伤来自胞嘧啶脱氨作用在降解样本中已有充分记录，例如来自古代和法医背景的样本。本研究检查了 DNA 修复处理对来自老年和降解骨骼样本的线粒体 DNA (mtDNA) 的影响。对 21 个非概率降解骨骼样本（年龄 50-70 岁）的 DNA 提取物进行了分析。对每个样本提取物的一部分使用商业修复试剂盒，即新英格兰生物实验室的 NEBNext FFPE DNA 修复试剂盒（马萨诸塞州伊普斯威奇）进行 DNA 修复。使用未处理和修复的 DNA 的平行实验，通过 PCR 和靶向捕获来富集 mtDNA。使用传统（Sanger 型；STS）和下一代测序（NGS）方法进行测序。虽然在 mtDNA 序列数据中可以观察到胞嘧啶脱氨作用，但观察到的损伤碱基水平因测序方法以及富集类型而异。STS PCR 扩增子数据未显示出可在未处理或修复样本集中从背景信号中区分出来的胞嘧啶脱氨作用的证据。然而，相同的 PCR 扩增子显示出 850 个 C ⁇ → T/G ⁇ → A 取代，与胞嘧啶脱氨作用一致，当使用 NGS 方法测序时，变异频率 (VF) 高达 25%。在修复后，NGS 扩增子数据中的碱基错配发生率因胞嘧啶脱氨作用而降低了 98%（至 10）。NGS 捕获数据表明 mtDNA 片段中的胞嘧啶脱氨作用水平较低（1-2%），DNA 修复可有效减轻这种情况。PCR 和捕获富集方法中胞嘧啶脱氨作用水平的观察差异可归因于所采用的 PCR 富集技术（例如低模板输入、增加 PCR 循环）的随机效应更大的倾向。总而言之，这些结果表明，当使用 NGS 方法对来自降解法医案例样本的 PCR 扩增 DNA 进行测序时，可能需要进行 DNA 修复。
