Verogen Inc., San Diego, CA 92121, USA.
Genes (Basel). 2021 Apr 19;12(4):599. doi: 10.3390/genes12040599.
Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing of multiple samples (libraries) and increased operational efficiencies. This report describes the design and developmental validation, according to forensic quality assurance standards, of end-to-end workflows for two multiplexes, comprised of ForenSeq mtDNA control region and mtDNA whole-genome kits the MiSeq FGx instrument and ForenSeq universal analysis software (UAS) 2.0/2.1. Polymerase chain reaction (PCR) enrichment and a tiled amplicon approach target small, overlapping amplicons (60-150 bp and 60-209 bp for the control region and mtGenome, respectively). The system provides convenient access to data files that can be used outside of the UAS if desired. Studies assessed a range of environmental and situational variables, including but not limited to buccal samples, rootless hairs, dental and skeletal remains, concordance of control region typing between the two multiplexes and as compared to orthogonal data, assorted sensitivity studies, two-person DNA mixtures and PCR-based performance testing. Limitations of the system and implementation considerations are discussed. Data indicated that the two mtDNA multiplexes, MiSeq FGx and ForenSeq software, meet or exceed forensic DNA quality assurance (QA) guidelines with robust, reproducible performance on samples of various quantities and qualities.
基于新一代测序（NGS），即高通量测序（MPS）的法医线粒体 DNA（mtDNA）分析与 Sanger 测序相比具有现代优势，例如每个碱基的深度覆盖（此处称为每个碱基对的读深度（bp））、同时对多个样本（文库）进行测序以及提高操作效率。本报告描述了根据法医质量保证标准，对两个多重扩增试剂盒的端到端工作流程的设计和开发验证，这两个试剂盒由 ForenSeq mtDNA 控制区和 mtDNA 全基因组试剂盒、MiSeq FGx 仪器和 ForenSeq 通用分析软件（UAS）2.0/2.1 组成。聚合酶链反应（PCR）富集和覆盖式扩增方法靶向小的重叠扩增子（控制区和 mtGenome 分别为 60-150bp 和 60-209bp）。该系统提供了方便访问数据文件的途径，如果需要，这些文件可以在 UAS 之外使用。研究评估了一系列环境和情境变量，包括但不限于口腔样本、无根毛发、牙齿和骨骼遗骸、两个多重扩增试剂盒的控制区分型一致性以及与正交数据的比较、各种灵敏度研究、两人混合 DNA 和基于 PCR 的性能测试。讨论了系统的局限性和实施注意事项。数据表明，两个 mtDNA 多重扩增试剂盒，MiSeq FGx 和 ForenSeq 软件，在各种数量和质量的样本上具有稳健、可重复的性能，满足或超过法医 DNA 质量保证（QA）指南的要求。
