Bennett Lindsay, Oldoni Fabio, Long Kelly, Cisana Selena, Madella Katrina, Wootton Sharon, Chang Joseph, Hasegawa Ryo, Lagacé Robert, Kidd Kenneth K, Podini Daniele
Metro Nashville Police Department Crime Laboratory, 400 Myatt Drive, Madison, TN, 37115, USA.
The Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, D.C., 20007, USA.
Int J Legal Med. 2019 May;133(3):719-729. doi: 10.1007/s00414-019-02010-7. Epub 2019 Feb 13.
Short tandem repeat polymorphisms (STRs) are the standard markers for forensic human identification. STRs are highly polymorphic loci analyzed using a direct PCR-to-CE (capillary electrophoresis) approach. However, STRs have limitations particularly when dealing with complex mixtures. These include slippage of the polymerase during amplification causing stutter fragments that can be indistinguishable from minor contributor alleles, preferential amplification of shorter alleles, and limited number of loci that can be effectively co-amplified with CE. Massively parallel sequencing (MPS), by enabling a higher level of multiplexing and actual sequencing of the DNA, provides forensic practitioners an increased power of discrimination offered by the sequence of STR alleles and access to new sequence-based markers. Microhaplotypes (i.e., microhaps or MHs) are emerging multi-allelic loci of two or more SNPs within < 300 bp that are highly polymorphic, have alleles all of the same length, and do not generate stutter fragments. The growing number of loci described in the literature along with initial mixture investigations supports the potential for microhaps to aid in mixture interpretation and the purpose of this study was to demonstrate that practically. A panel of 36 microhaplotypes, selected from a set of over 130 loci, was tested with the Ion S5™ MPS platform (Thermo Fisher Scientific) on single-source samples, synthetic two-to-six person mixtures at different concentrations/contributor ratios, and on crime scene-like samples. The panel was tested both in multiplex with STRs and SNPs and individually. The analysis of single-source samples showed that the allele coverage ratio across all loci was 0.88 ± 0.08 which is in line with the peak height ratio of STR alleles in CE. In mixture studies, results showed that the input DNA can be much higher than with conventional CE, without the risk of oversaturating the detection system, enabling an increased sensitivity for the minor contributor in imbalanced mixtures with abundant amounts of DNA. Furthermore, the absence of stutter fragments simplifies the interpretation. On casework-like samples, MPS of MHs enabled the detection of a higher number of alleles from minor donors than MPS and CE of STRs. These results demonstrated that MPS of microhaplotypes can complement STRs and enhance human identification practices when dealing with complex imbalanced mixtures.
短串联重复多态性（STRs）是法医人类身份鉴定的标准标记。STRs是高度多态性位点，采用直接聚合酶链式反应-毛细管电泳（PCR-CE）方法进行分析。然而，STRs存在局限性，尤其是在处理复杂混合物时。这些局限性包括扩增过程中聚合酶的滑动导致拖尾片段，这些片段可能与次要贡献者等位基因无法区分、短等位基因的优先扩增以及可与CE有效共扩增的位点数量有限。大规模平行测序（MPS）通过实现更高水平的多重化和DNA的实际测序，为法医从业者提供了更高的鉴别能力，这得益于STR等位基因序列以及对新的基于序列的标记的获取。微单倍型（即微单倍型或MHs）是新兴的多等位基因位点，由<300 bp内的两个或更多单核苷酸多态性（SNP）组成，具有高度多态性，所有等位基因长度相同，且不会产生拖尾片段。文献中描述的位点数量不断增加以及初步的混合物研究支持了微单倍型有助于混合物解释的潜力，本研究的目的就是实际证明这一点。从130多个位点中选出的36个微单倍型组成的面板，在Ion S5™ MPS平台（赛默飞世尔科技公司）上对单源样本、不同浓度/贡献者比例的合成二至六人混合物以及类似犯罪现场的样本进行了测试。该面板既与STRs和SNP进行多重测试，也单独进行测试。单源样本分析表明，所有位点的等位基因覆盖率为0.88±0.08，这与CE中STR等位基因的峰高比一致。在混合物研究中，结果表明输入的DNA量可比传统CE高得多，且不存在检测系统饱和的风险，从而提高了对DNA含量丰富的不平衡混合物中次要贡献者的检测灵敏度。此外，拖尾片段的缺失简化了解释。在类似案件样本上，微单倍型的MPS能够比STRs的MPS和CE检测到更多来自次要供体的等位基因。这些结果表明，在处理复杂的不平衡混合物时，微单倍型的MPS可以补充STRs并增强人类身份鉴定实践。
