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首页> 外文期刊>International journal of legal medicine >Mixture deconvolution by massively parallel sequencing of microhaplotypes
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Mixture deconvolution by massively parallel sequencing of microhaplotypes

机译：通过微生物型的大规模平行测序混合去折叠

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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 (TM) 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.
机译：短串联重复多态性（strs）是法医人体鉴定的标准标记。使用直接PCR-TO-CE（毛细管电泳）方法分析STRS高度多态性基因座。然而，在处理复杂的混合物时，strs特别有局限性。这些包括在扩增期间的聚合酶的滑点，导致口吃片段可以与次要贡献者等位基因中难以区分，优先扩增等位基因的优先扩增，并且可以用CE有效地共同扩增有限数量的基因座。通过实现更高水平的DNA多路复用和实际测序的大规模平行测序（MPS），提供了法医从业者通过STR等位基因序列提供的歧视力增加，并获得新的基于序列的标记。微生物（即微宝或MHS）在<300bp中产生的两个或更多个SNP的多等位基因座在高度多态性中，具有相同长度的等位基因，并且不会产生油断裂片段。文献中描述的越来越多的基因座以及初始混合性调查支持微宝的可能性，以帮助混合解释，本研究的目的是实际证明。在单源样本上使用离子S5（TM）MPS平台（Thermo Fisher Scientific）测试了36个微生型，选自超过130个基因座的微生型，以单源样本，在不同浓度/贡献者的合成二到六个人混合物中进行测试比率，以及犯罪现场样本。该面板在与STRS和SNP中的多路复用中进行测试，并单独测试。单源样本的分析表明，所有基因座的等位基因覆盖率为0.88 +/- 0.08，其符合Ce中的str等位基因的峰值高度比。在混合研究中，结果表明，输入DNA可以远高于常规CE，而不会过度冒失检测系统的风险，从而使得在具有丰富量的DNA中的不平衡混合物中的轻微贡献者的敏感性。此外，不存在口号片段简化了解释。在案例样本样本上，MPS的MPS使得检测来自小于MPS和STR的MPS和CE的较小捐助者的等位基因。这些结果表明，MPS的MICSPLOTYPES可以在处理复杂的不平衡混合物时增强人类识别实践。

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