A mixed DNA profile controversy revisited

Semaan et al. (J Forensic Res, 2020, 11, 453) discuss a mock case “where eight different individuals [P₁ through P₈] could not be excluded in a mixed DNA analysis. Even though … expert DNA mixture analysis software was used.” Two of these are the true donors. The LRs reported are incorrect due to the incorrect entry of propositions into LRmix Studio. This forced the software to account for most of the alleles as drop-in, resulting in LRs 60–70 orders of magnitude larger than expected. P₁, P₂, P₄, P₅, and P₈ can be manually excluded using peak heights. This has relevance when using LRmix which does not use peak heights. We extend the work using the same two reference genotypes who were the true contributors as Semaan et al. (J Forensic Res, 2020, 11, 453). We simulate three two-donor mixtures with peak heights using these two genotypes and analyze using STRmix?. For the simulated 1:1 mixture, one of the non-donors’ LRs supported him being a contributor when no conditioning was used. When considered in combination with any other potential donors (i.e., with conditioning), this non-donor was correctly eliminated. For the 3:1 mixture, all results correctly supported that the non-donors were not contributors. The low-template 4:1 mixture LRs with no conditioning showed support for all eight profiles as donors. However, the results from pair-wise conditioning showed that only the two ground truth donors had LRs supporting that they were contributors to the mixture. We recommend the use of peak heights and conditioning profiles, as this allows better sensitivity and specificity even when the persons share many alleles.     

HLA基因分型技术的进展与展望

研究人的主要组织相容性复合物HLA(humanleucocyteantigen,人类白细胞抗原)的结构和功能有助于认识生命现象的本质以及疾病的致病机理,因此长期以来一直是生命科学研究的重点之一。随着分子生物学技术的迅速发展,HLA分型已经由传统的血清学水平向DNA水平过渡,并取得了很好的效果。本文对目前各种HLA基因分型方法的原理和优缺点结合自己的应用经验做了评价,并简要介绍了在HLA基因分型研究中出现的新的动态。     

TrueAllele® Genotype Identification on DNA Mixtures Containing up to Five Unknown Contributors

Computer methods have been developed for mathematically interpreting mixed and low‐template DNA. The genotype modeling approach computationally separates out the contributors to a mixture, with uncertainty represented through probability. Comparison of inferred genotypes calculates a likelihood ratio (LR), which measures identification information. This study statistically examined the genotype modeling performance of Cybergenetics TrueAllele^® computer system. High‐ and low‐template DNA mixtures of known randomized composition containing 2, 3, 4, and 5 contributors were tested. Sensitivity, specificity, and reproducibility were established through LR quantification in each of these eight groups. Covariance analysis found LR behavior to be relatively invariant to DNA amount or contributor number. Analysis of variance found that consistent solutions were produced, once a sufficient number of contributors were considered. This study demonstrates the reliability of TrueAllele interpretation on complex DNA mixtures of representative casework composition. The results can help predict an information outcome for a DNA mixture analysis.     

SMART:自主研发的混合STR图谱分析系统

目的混合STR图谱的分析是法医遗传学领域的一个难题。目前国内大多数法医DNA实验室依靠人工方法分析混合STR图谱,费时费力,且拆分效果也多有主观性,难以满足日益增长的混合STR图谱分析的需求。本文介绍一套自主研发的混合STR图谱分析系统——SMART(STR Mixture Analysis and Resolution Tools),其能够实现对混合STR图谱的自动化分析,包括拆分混合STR图谱和计算似然比。方法SMART使用的STR峰高的概率模型,考虑了影子峰、降解、基因座特异性扩增效率、峰高变异、插入峰、峰丢失等因素。模型通过比较各个基因型集合拟合当前混合STR图谱的似然值大小来推断最有可能的基因型集合,采用了马尔可夫链蒙特卡洛(MCMC)算法进行求解。结果SMART可应用于不同试剂盒和遗传分析仪产生的数据。经使用ABI-3500XL遗传分析仪和Global Filer试剂盒测试,SMART能够实现对2~5人的混合STR图谱的分析,输出包括混合比例、混合图谱质量、各个贡献者单人的STR基因分型、似然比等多项结果。结论SMART作为一款自主研发的混合STR图谱分析系统,其基本涵盖了国外同类分析系统所具有的功能,技术性能指标达到国际先进水平,能够满足一线法医工作者对于混合STR图谱分析的多样化需求,提高混合STR图谱结果的利用率。     

The Probabilistic Genotyping Software STRmix: Utility and Evidence for its Validity

Forensic DNA interpretation is transitioning from manual interpretation based usually on binary decision‐making toward computer‐based systems that model the probability of the profile given different explanations for it, termed probabilistic genotyping (PG). Decision‐making by laboratories to implement probability‐based interpretation should be based on scientific principles for validity and information that supports its utility, such as criteria to support admissibility. The principles behind STRmix? are outlined in this study and include standard mathematics and modeling of peak heights and variability in those heights. All PG methods generate a likelihood ratio (LR) and require the formulation of propositions. Principles underpinning formulations of propositions include the identification of reasonably assumed contributors. Substantial data have been produced that support precision, error rate, and reliability of PG, and in particular, STRmix?. A current issue is access to the code and quality processes used while coding. There are substantial data that describe the performance, strengths, and limitations of STRmix?, one of the available PG software.     

激光捕获显微分离技术在DNA检验中的应用研究

目的建立一套显微细胞捕获技术用于法医学生物检材DNA检验方法。方法使用VeritasTM LCM激光捕获仪,采用紫外加红外的捕获方式,对框架覆膜玻片上经苏木素染色口腔上皮细胞进行捕获,采用改良硅珠法提取细胞DNA,使用Identifiler TM试剂盒在5μL体系中进行PCR扩增。结果成功获得20个口腔上皮细胞的13个以上完整STR基因座分型谱带。结论本研究建立的方法适合法医学生物检材制成的染色涂片上细胞的DNA检验。     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

Using the Nondonor Distribution to Improve Communication and Inform Decision Making for Low LRs from Minor Contributors in Mixed DNA Profiles

The reporting of a likelihood ratio (LR) calculated from probabilistic genotyping software has become more popular since 2015 and has allowed for the use of more complex mixtures at court. The meaning of “inconclusive” LRs and how to communicate the significance of low LRs at court is now important. We present a method here using the distribution of LRs obtained from nondonors. The nondonor distribution is useful for examining calibration and discrimination for profiles that have produced LRs less than about 10⁴. In this paper, a range of mixed DNA profiles of varying quantity were constructed and the LR distribution considering the minor contributor for a number of nondonors was compared to the expectation given a calibrated system. It is demonstrated that conditioning genotypes should be used where reasonable given the background information to decrease the rate of nondonor LRs above 1. In all 17 cases examined, the LR for the minor donor was higher than the nondonor LRs, and in 12 of the 17 cases, the 99.9 percentile of the nondonor distribution was lower when appropriate conditioning information was used. The output of the tool is a graph that can show the position of the LR for the person of interest set against the nondonor LR distribution. This may assist communication between scientists and the court.     

Validating TrueAllele® Interpretation of DNA Mixtures Containing up to Ten Unknown Contributors

Most DNA evidence is a mixture of two or more people. Cybergenetics TrueAllele^® system uses Bayesian computing to separate genotypes from mixture data and compare genotypes to calculate likelihood ratio (LR) match statistics. This validation study examined the reliability of TrueAllele computing on laboratory-generated DNA mixtures containing up to ten unknown contributors. Using log(LR) match information, the study measured sensitivity, specificity, and reproducibility. These reliability metrics were assessed under different conditions, including varying the number of assumed contributors, statistical sampling duration, and setting known genotypes. The main determiner of match information and variability was how much DNA a person contributed to a mixture. Observed contributor number based on data peaks gave better results than the number known from experimental design. The study found that TrueAllele is a reliable method for analyzing DNA mixtures containing up to ten unknown contributors.     

Significant association of TH01 allele 9.3 and SIDS

Sudden infant death syndrome (SIDS) constitutes a considerable percentage of infant death of unknown etiology. Individual catecholamine response variation is suspected to play a role in SIDS. TH01 is a tetrameric short tandem repeat marker in the tyrosine hydroxylase gene, which regulates gene expression and catecholamine production with allele 9.3 exerting a particularly strong effect on noradrenaline production. We investigated in an age-controlled study the TH01 allele frequencies in 127 cases of SIDS and 406 control cases to assess whether in SIDS cases a distinct TH01 allele distribution could be determined as has been reported by a previous study. We found that genotypes containing one or two 9.3 alleles were significantly more frequent in SIDS patients (58.2%) than in control subjects (48.4%, p=0.038), whereas all other alleles were more frequent in the control subjects. Our findings support the notion that there exists a significant association between TH01 gene configuration and SIDS.