National Research Institute of Police Science, Chiba 277-0882, Japan.
National Research Institute of Police Science, Chiba 277-0882, Japan.
Forensic Sci Int Genet. 2022 Jan;56:102624. doi: 10.1016/j.fsigen.2021.102624. Epub 2021 Oct 29.
Saliva samples obtained from crime scenes often contain body fluids from other people, which makes it difficult to not only interpret the obtained DNA profiles, but also interpret saliva identification test results. α-amylase activity, an indicator of most saliva identification methods, can be slightly detected in other body fluids. This study aimed to overcome these difficulties. Here, we identified 13 saliva-specific methylated regions and five saliva-specific unmethylated regions neighboring common single nucleotide polymorphisms (SNPs) by array-based genome-wide methylation analysis of pooled saliva, blood, semen, or vaginal swab samples. Bisulfite sequencing by massively parallel sequencing (MPS) technology was then performed using individual body fluid samples to evaluate the saliva-specificity of each CpG of the three regions selected from the identified candidates. Although no single CpG demonstrated complete saliva-specificity, we found that the reads that were simultaneously (un)methylated at the selected neighboring two to three CpGs of each region were highly specific for saliva DNA. Based on these findings, we then designed MPS-based bisulfite sequencing assays for each region to analyze the selected CpGs and SNP(s) on the same read. These assays could identify the saliva of a target person from body fluid mixtures of known contributors (individual-specific saliva identification) by calculating the ratios of simultaneous (un)methylation at the selected CpGs within the reads containing SNP alleles unique to the target person. Moreover, these assays could indicate the SNP types of saliva DNA (saliva-specific genotyping) from body fluid mixtures by analyzing the alleles of the reads simultaneously (un)methylated at the selected CpGs, while careful attention should be paid to interpret the results of heterologous genotypes. Although further regions should be identified, especially for saliva-specific individual identification, the CpG-SNP approach may be an effective method to interpret the complicated results obtained from saliva-containing body fluid mixtures.
从犯罪现场获得的唾液样本通常包含其他人的体液，这不仅使得解释获得的 DNA 图谱变得困难，而且还使得解释唾液鉴定测试结果变得困难。大多数唾液鉴定方法的指标α-淀粉酶活性，在其他体液中也能被轻微检测到。本研究旨在克服这些困难。在这里，我们通过对 pooled saliva、blood、semen 或 vaginal swab 样本进行基于阵列的全基因组甲基化分析，鉴定出 13 个唾液特异性甲基化区域和 5 个紧邻常见单核苷酸多态性（SNP）的唾液特异性非甲基化区域。然后使用个体体液样本通过大规模平行测序（MPS）技术进行亚硫酸氢盐测序，以评估从鉴定出的候选者中选择的三个区域的每个 CpG 的唾液特异性。尽管没有一个 CpG 表现出完全的唾液特异性，但我们发现，在所选择的每个区域的两个到三个相邻 CpG 同时（未）甲基化的reads 对唾液 DNA 具有高度特异性。基于这些发现，我们随后为每个区域设计了基于 MPS 的亚硫酸氢盐测序检测，以分析选定的 CpG 和 SNP（s）在同一 read 上。这些检测可以通过计算包含目标个体特有的 SNP 等位基因的 read 中选定 CpG 同时（未）甲基化的比率，从已知供体的体液混合物中鉴定出目标个体的唾液（个体特异性唾液鉴定）。此外，这些检测可以通过分析选定 CpG 同时（未）甲基化的 read 的等位基因，从体液混合物中指示唾液 DNA 的 SNP 类型（唾液特异性基因分型），但应谨慎注意解释异质基因型的结果。虽然应该进一步鉴定其他区域，特别是用于唾液特异性个体鉴定，但 CpG-SNP 方法可能是解释含有唾液的体液混合物中获得的复杂结果的有效方法。
