Fordyce Sarah L, Mogensen Helle Smidt, Børsting Claus, Lagacé Robert E, Chang Chien-Wei, Rajagopalan Narasimhan, Morling Niels
Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Thermo Fisher Scientific, South San Francisco, CA, USA.
Forensic Sci Int Genet. 2015 Jan;14:132-40. doi: 10.1016/j.fsigen.2014.09.020. Epub 2014 Oct 5.
Second-generation sequencing (SGS) using Roche/454 and Illumina platforms has proved capable of sequencing the majority of the key forensic genetic STR systems. Given that Roche has announced that the 454 platforms will no longer be supported from 2015, focus should now be shifted to competing SGS platforms, such as the MiSeq (Illumina) and the Ion Personal Genome Machine (Ion PGM™; Thermo Fisher). There are currently several challenges faced with amplicon-based SGS STR typing in forensic genetics, including current lengths of amplicons for CE-typing and lack of uniform data analysis between laboratories. Thermo Fisher has designed a human identification (HID) short tandem repeat (STR) 10-plex panel including amelogenin, CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX and vWA, where the primers have been designed specifically for the purpose of SGS and the data analysis is supported by Ion Torrent™ software. Hence, the combination of the STR 10-plex and the Ion PGM™ represents the first fully integrated SGS STR typing solution from PCR to data analysis. In this study, four experiments were performed to evaluate the alpha-version of the STR 10-plex: (1) typing of control samples; (2) analysis of sensitivity; (3) typing of mixtures; and (4) typing of biological crime case samples. Full profiles and concordant results between replicate SGS runs and CE-typing were observed for all control samples. Full profiles were seen with DNA input down to 50 pg, with the exception of a single locus drop-out in one of the 100 pg dilutions. Mixtures were easily deconvoluted down to 20:1, although alleles from the minor contributor had to be identified manually as some signals were not called by the Ion Torrent™ software. Interestingly, full profiles were obtained for all biological samples from real crime and identification cases, in which only partial profiles were obtained with PCR-CE assays. In conclusion, the Ion Torrent™ HID STR 10-plex panel offers an all-in-one solution from amplification of STRs and amelogenin, and sequencing to data analysis.
事实证明，使用罗氏/454和Illumina平台的第二代测序（SGS）能够对大多数关键法医遗传学短串联重复序列（STR）系统进行测序。鉴于罗氏已宣布自2015年起不再支持454平台，现在应将重点转移到其他竞争的SGS平台上，如MiSeq（Illumina）和Ion个人基因组测序仪（Ion PGM™；赛默飞世尔科技公司）。目前，法医遗传学中基于扩增子的SGS STR分型面临着若干挑战，包括当前用于毛细管电泳（CE）分型的扩增子长度以及实验室之间缺乏统一的数据分析。赛默飞世尔科技公司设计了一种人类身份识别（HID）短串联重复序列（STR）十重检测试剂盒，包含牙釉蛋白、CSF1PO、D16S539、D3S1358、D5S818、D7S820、D8S1179、TH01、TPOX和vWA，其引物是专门为SGS目的设计的，并且数据分析由Ion Torrent™软件提供支持。因此，STR十重检测试剂盒与Ion PGM™的组合代表了首个从聚合酶链式反应（PCR）到数据分析的完全集成的SGS STR分型解决方案。在本研究中，进行了四项实验来评估STR十重检测试剂盒的alpha版本：（1）对照样本分型；（2）灵敏度分析；（3）混合样本分型；以及（4）生物犯罪案件样本分型。所有对照样本在重复的SGS运行和CE分型之间均观察到完整的图谱和一致的结果。DNA输入低至50皮克时可看到完整的图谱，但在100皮克稀释样本中的一个出现了单个位点缺失。混合样本可轻松解卷积至20:1，不过次要贡献者的等位基因必须手动识别，因为Ion Torrent™软件未调用某些信号。有趣的是，从实际犯罪和身份识别案件中获取的所有生物样本均获得了完整的图谱，而在这些案件中，PCR-CE检测仅获得了部分图谱。总之，Ion Torrent™ HID STR十重检测试剂盒提供了一个从STR和牙釉蛋白的扩增、测序到数据分析的一体化解决方案。
