van der Gaag Kristiaan J, de Leeuw Rick H, Hoogenboom Jerry, Patel Jaynish, Storts Douglas R, Laros Jeroen F J, de Knijff Peter
Forensic Laboratory for DNA Research, Department of Human Genetics, Leiden University Medical Centre, Postzone S 05 P, P.O. Box 9600, 2300 RC Leiden, The Netherlands; Biological Traces, Netherlands Forensic Institute, Laan van Ypenburg 6, 2497GB The Hague, The Netherlands.
Forensic Laboratory for DNA Research, Department of Human Genetics, Leiden University Medical Centre, Postzone S 05 P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Forensic Sci Int Genet. 2016 Sep;24:86-96. doi: 10.1016/j.fsigen.2016.05.016. Epub 2016 Jun 7.
Current forensic DNA analysis predominantly involves identification of human donors by analysis of short tandem repeats (STRs) using Capillary Electrophoresis (CE). Recent developments in Massively Parallel Sequencing (MPS) technologies offer new possibilities in analysis of STRs since they might overcome some of the limitations of CE analysis. In this study 17 STRs and Amelogenin were sequenced in high coverage using a prototype version of the Promega PowerSeq™ system for 297 population samples from the Netherlands, Nepal, Bhutan and Central African Pygmies. In addition, 45 two-person mixtures with different minor contributions down to 1% were analysed to investigate the performance of this system for mixed samples. Regarding fragment length, complete concordance between the MPS and CE-based data was found, marking the reliability of MPS PowerSeq™ system. As expected, MPS presented a broader allele range and higher power of discrimination and exclusion rate. The high coverage sequencing data were used to determine stutter characteristics for all loci and stutter ratios were compared to CE data. The separation of alleles with the same length but exhibiting different stutter ratios lowers the overall variation in stutter ratio and helps in differentiation of stutters from genuine alleles in mixed samples. All alleles of the minor contributors were detected in the sequence reads even for the 1% contributions, but analysis of mixtures below 5% without prior information of the mixture ratio is complicated by PCR and sequencing artefacts.
当前的法医DNA分析主要通过使用毛细管电泳（CE）分析短串联重复序列（STR）来鉴定人类供体。大规模平行测序（MPS）技术的最新发展为STR分析提供了新的可能性，因为它们可能克服CE分析的一些局限性。在本研究中，使用Promega PowerSeq™系统的原型版本对来自荷兰、尼泊尔、不丹和中非俾格米人的297份群体样本进行了高覆盖度的17个STR和牙釉蛋白基因测序。此外，分析了45个不同程度的两人混合样本，其中次要贡献低至1%，以研究该系统对混合样本的性能。关于片段长度，发现MPS和基于CE的数据完全一致，这标志着MPS PowerSeq™系统的可靠性。正如预期的那样，MPS呈现出更宽的等位基因范围、更高的鉴别力和排除率。高覆盖度测序数据用于确定所有位点的拖尾特征，并将拖尾比率与CE数据进行比较。分离长度相同但拖尾比率不同的等位基因可降低拖尾比率的总体变化，并有助于在混合样本中将拖尾与真实等位基因区分开来。即使次要贡献者的贡献低至1%，其所有等位基因也能在序列读数中检测到，但对于低于5%的混合样本，如果没有混合比率的先验信息，PCR和测序假象会使分析变得复杂。
