Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain.
Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain.
Forensic Sci Int Genet. 2023 Nov;67:102937. doi: 10.1016/j.fsigen.2023.102937. Epub 2023 Sep 28.
We have adapted an established Ampliseq microhaplotype panel for nanopore sequencing with the Oxford Nanopore Technologies (ONT) system, as a cost-effective and highly scalable solution for forensic genetics applications. For this purpose, we designed a protocol combining direct PCR amplification from unextracted DNA with ONT library construction and sequencing using the MinION device and workflow. The analysis of reference samples at input amounts of 5-10 ng of DNA demonstrates stable coverage patterns, allele balance, and strand bias, reaching profile completeness and concordance rates of ∼95%. Similar levels were achieved when using direct-PCR from blood, buccal and semen swabs. Dilution series results indicate sensitivity is maintained down to 250 pg of input DNA, and informative profiles are produced down to 62.5 pg. Finally, we demonstrated the forensic utility of the nanopore workflow by analyzing two third degree pedigrees that showed low likelihood ratio values after the analysis of an extended panel of 38 STRs, achieving likelihood ratios 2-3 orders of magnitude higher when testing with the MinION-based haplotype data.
我们针对牛津纳米孔技术（ONT）系统，对已建立的 Ampliseq 微单倍型面板进行了适配，使其成为法医遗传学应用中一种具有成本效益且高度可扩展的解决方案。为此，我们设计了一个方案，将直接从未提取的 DNA 进行 PCR 扩增与 ONT 文库构建和 MinION 设备及工作流程测序相结合。在输入量为 5-10ng DNA 的参考样本分析中，我们证明了该方案具有稳定的覆盖模式、等位基因平衡和链偏倚，达到了约 95%的完整度和一致性率。使用血液、口腔和精液拭子的直接-PCR 也可实现类似水平。稀释系列结果表明，该方案的灵敏度可维持在 250pg 的输入 DNA 水平，并且在输入 DNA 水平低至 62.5pg 时也能产生信息丰富的图谱。最后，我们通过分析两个三亲系谱证明了纳米孔工作流程的法医学实用性，在对 38 个 STR 进行扩展面板分析后，这些系谱显示出低似然比值，而使用基于 MinION 的单倍型数据进行测试时，似然比可提高 2-3 个数量级。
