基于下一代测序的法医DNA疑难检材全解析度STR检验探索研究

STR genotyping, accepted as the dominant technology in the field of forensic DNA, is widely used in solving criminal cases and disaster victim identification. Based on the capillary electrophoresis platform, the forensic DNA community has been doing well in STR genotyping of the routine biological samples. However, further efforts are required for examining the trace DNA, degraded DNA and mixed DNA samples, which are frequently encountered in criminal cases as challenging forensic DNA samples. Next generation sequencing (NGS) technology has the potential to solve such problems. Furthermore, NGS is capable of providing full resolution STR data at the sequence variation level, which could significantly improve the individual identification capability of the forensic STR loci. Here the project focuses on NGS-based STR genotyping of the forensic trace DNA, degraded DNA and mixed DNA samples. Employing NGS, real-time PCR and capillary electrophoresis as major research techniques, the project is divided into three sections, which are (1) NGS and CE performance comparison on examination of the challenging forensic DNA samples and optimization of the NGS library preparation procedures for challenging samples; (2) full resolution STR data and forensic applications; and (3) research on the correlation between NGS sequencing depth and STR data accuracy. Systematic research will be carried out to evaluate the sensitivity, DNA degradation tolerance and mixed sample discriminative capacity of the NGS platform on STR genotyping. By the research work described above, the project aims to establish the strategy for NGS-based full resolution STR genotyping of challenging forensic DNA samples, improving the capabilities for forensic biological evidence examination and STR sequence variation information utilization.

STR分型是法医DNA领域的主流技术，在刑事案件侦查举证、灾害遇难者身份认定等领域具有广泛应用。基于毛细管电泳平台，法医DNA学界已经可以解决各类常规检材的DNA检验问题，但对于刑事案件中经常遇到的微量、降解、混合等疑难生物检材，检验能力仍有待提高。下一代测序（NGS）技术具备解决以上问题的潜力，且提供序列多态水平的全解析度STR数据，可显著提升STR基因座的个体识别效力。本项目围绕微量、降解、混合等三类法医疑难检材检验问题，以NGS、荧光定量PCR、毛细管电泳为主要技术手段，从NGS与毛细管电泳平台对疑难检材检验能力比较和文库构建方法优化、全解析度STR数据研究与法庭科学应用、测序深度与数据准确度关联性三个环节入手，系统探究NGS在灵敏度、降解容忍度、混合检材区分度等方面的检验能力，建立基于NGS平台STR分型的法医疑难检材检验策略，提高法医生物物证检验鉴定和STR序列多态信息利用能力。

STR分型是法医DNA领域的主流技术，但对于刑事案件中经常遇到的微量、降解、混合等疑难生物检材，检验能力仍有待提高。下一代测序（NGS）技术具备解决以上问题的潜力，且提供序列多态水平的全解析度STR数据，可显著提升STR基因座的个体识别效力。本项目围绕微量、降解、混合等三类法医疑难检材检验问题，从NGS与毛细管电泳平台对疑难检材检验能力比较和文库构建方法优化、全解析度STR数据研究与法庭科学应用、测序深度与数据准确度关联性三个环节入手，建立了微量DNA文库构建方法，测得NGS-STR技术的灵敏度低至62pg基因组DNA，可从降解指数为8.947的样本中获得完整分型，可从1：9和9：1比例的混合样本中获得微量组分正确分型。本项目积累了宝贵的基于序列的中国汉族人群STR遗传多态性基础数据，基于数学模型和真实人群数据分别计算了法庭科学参数，量化评估结果表明序列多态STR的累积随机匹配概率相比长度多态STR可至少降解2个数量级。本项目系统评估了测序深度对实验结果准确性的影响，提出了将最低测序深度与平均测序深度的比值作为设定数据分析阈值的重要参考指标，提高了法医生物物证检验鉴定和STR序列多态信息利用能力。