Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States; University of California, Davis, Forensic Science Graduate Program, 1909 Galileo Ct. Ste. B, Davis CA 95618, United States.
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, United States; UCSF Benioff Children's Hospital Oakland, Hematology/Oncology Department 747 52nd St, Oakland, CA 94609, United States.
Forensic Sci Int Genet. 2018 May;34:186-196. doi: 10.1016/j.fsigen.2018.01.010. Epub 2018 Feb 4.
DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1 ng and 10 ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10 ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples.
从法医学样本中提取的 DNA 可能高度碎片化，且数量有限。当 DNA 高度碎片化时，基于常规聚合酶链反应（PCR）的短串联重复序列（STR）分析可能会失败，因为引物结合位点可能不存在于单个模板分子上。单核苷酸多态性（SNP）可以作为分析降解样本的另一种遗传标记，因为目标变异是单个碱基。然而，基于常规 PCR 的 SNP 分析方法仍然需要完整的引物结合位点来进行靶标扩增。最近，针对目标物的探针捕获方法在使用下一代测序（NGS）技术对降解和古老骨样本进行靶向富集方面取得了成功。本研究的目的是设计并测试一种针对法医相关核 SNP 标记的探针捕获分析方法，用于对降解和有限量 DNA 样本以及混合物进行克隆和大规模平行测序（MPS）。选择了一组总计 451 个核 SNP（375 个 SNP 和 36 个微单倍型标记）的 411 个多态性标记用于定制探针捕获面板。这些 SNP 标记是为广泛的法医应用选择的，包括个体识别、亲属关系和谱系分析以及混合物分析。通过分析 15 个样本在 25ng 输入 DNA 时的读深度和杂合子等位基因平衡来表征定制 SNP 探针捕获 NGS 分析的性能。根据观察到的 451 个 SNP 中的 426 个 SNP 的读深度≥500X 和杂合子等位基因平衡在±10%偏差范围内（50:50）来建立性能阈值。这 426 个 SNP 分析了大小选择样本（在≤75bp、≤100bp、≤150bp、≤200bp 和≤250bp）和模拟降解样本（平均片段化至 150bp）。选择的≤75bp 样本在不同 DNA 量和低至 0.5ng 时表现出 99-100%可报告 SNP。模拟降解样本在 1ng 和 10ng 时表现出>90%可报告 SNP。最后，在 10ng 的贡献者不同比例下测试了两人男性混合样本。总体而言，在所有混合比例下，观察到 85-100%的等位基因是次要贡献者特有的。这些使用 SNP 探针捕获 NGS 系统的研究结果证明了该系统在法医相关降解和混合 DNA 样本中的应用具有概念验证意义。
