Forensic interpretation of Y-chromosomal DNA mixtures

The mathematical concept previously introduced for the forensic interpretation of DNA mixtures using non-associated genetic markers has been adapted to the assessment of haplotypes. Such calculus is required, for example, when Y-chromosomal markers are used in forensics. In addition to outlining the general mathematical framework, we devise two approaches to its practical computational implementation, involving either the inclusion-exclusion principle of probability theory or a recursion in the number of unknown contributors invoked. The two approaches scale differently, depending upon the complexity of the case and the diversity of the markers used. The performance of Y-chromosomal microsatellites (Y-STRs) as a means of trace donor discrimination has been assessed by simulation, using the derived formulas. Based upon data from the Y-chromosomal Haplotype Reference Database (YHRD), the exclusion chance of a non-contributor is shown to vary between 95% in the case of two contributors, and 70% for five contributors. With only one additional contributor, half of all contributing suspects would yield a log-likelihood ratio in favour of donorship of 1.61 or higher, although the median drops to 0.66 with four additional contributors. It must be emphasised that these estimates of the discriminatory power of Y-STRs are likely to be conservative since the simulations involved only haplotypes known to occur in YHRD.     

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.     

混合斑的研究进展

混合斑组成复杂,长期以来是法医物证研究的难题之一。混合斑主要分为两类:一是性侵害案件中大量女性阴道上皮细胞混合少量精子;二是普通来源的生物检材之间的混合,如血-血、唾液-血液混合等。近些年来,对混合斑的研究日渐深入,技术层面或理论分析都更加客观和多元化。针对第一类混合斑,出现了荧光/磁性激活、显微操纵、声波差异分离法等分离精子的新技术;第二类混合斑是研究的难点,近年来大规模平行测序、液滴微流控技术、乳液PCR(e PCR)、全基因组扩增等技术的出现,提高了次要成分的检出机率。此外,遗传标记也有相应的发展,传统遗传标记常染色体STR、Y-STR、SNP等检测位点越来越丰富,新兴遗传标记DIP、DIP-STR、SNPSTR、mt DNA-SNP、微单倍型等在混合斑研究中发挥的作用初步显露出来。文章最后涉及混合斑分析理论和计算软件的最新发展。     

Exemption of chemical mixtures. Final rule

On December 15, 2004, the Drug Enforcement Administration (DEA) published a Final Rule corrected January 4, 2005) that implemented new regulations concerning chemical mixtures that contain any of the 27 listed chemicals. The Final Rule added a new provision not previously raised by DEA in any proposed rulemaking. This newly introduced provision exempted domestic and import transactions in chemical mixtures that are regulated solely due to the presence of the List II solvent chemicals acetone, ethyl ether, 2-butanone, or toluene from the Controlled Substances Act (CSA) recordkeeping and reporting requirements. Because this exemption was not previously proposed in any rulemaking, DEA implemented this exemption on an interim basis and requested public comment on this exemption provision. Based upon a review of all comments, DEA is finalizing this exemption. As such, domestic and import transactions in chemical mixtures containing the List II chemicals acetone, ethyl ether, 2-butanone, and toluene shall be exempt from CSA chemical recordkeeping and reporting requirements.     

Interpreting DNA mixtures in structured populations.

DNA profiles from multiple-contributor samples are interpreted by comparing the probabilities of the profiles under alternative propositions. The propositions may specify some known contributors to the sample and may also specify a number of unknown contributors. The probability of the alleles carried by the set of people, known or unknown, depends on the allelic frequencies and also upon any relationships among the people. Membership of the same subpopulation implies a relationship from a shared evolutionary history, and this effect has been incorporated into the probabilities. This acknowledgment of the effects of population structure requires account to be taken of all people in a subpopulation who are typed, whether or not they contributed to the sample.     

Likelihood ratios for complex mixtures with relatives

Likelihood ratios used for the analysis of complex DNA mixtures depend on a number of modeling assumptions and parameter estimates. In particular, the LR does not give information about the relative weight of the separate contributors for hypotheses conditioned on several contributors. An alternative is to evaluate the observed LR with respect to likelihood ratios expected under the defense hypothesis. Further, a p-value corresponding to the LR can be calculated. The p-value is the probability of observing a LR equally large or larger than the one observed, if the defense hypothesis is true. In this paper we investigate the distribution of likelihood ratios for mixtures with drop-in and drop-out and related contributors. Disregarding a plausible close relative of the suspect as an alternative contributor may overestimate the LR against a suspect.     

PENDULUM--a guideline-based approach to the interpretation of STR mixtures

Several years ago, a theory to interpret mixed DNA profiles was proposed that included a consideration of peak area using the method of least squares. This method of mixture interpretation has not been widely adopted because of the complexity of the associated calculations. Most reporting officers (RO) employ an experience and judgement based approach to the interpretation of mixed DNA profiles. Here we present an approach that has formalised the thinking behind this experience and judgement. This has been written into a computer program package called PENDULUM. The program uses a least squares method to estimate the pre-amplification mixture proportion for two potential contributors. It then calculates the heterozygous balance for all of the potential sets of genotypes. A list of "possible" genotypes is generated using a set of heuristic rules. External to the programme the candidate genotypes may then be used to formulate likelihood ratios (LR) that are based on alternative casework propositions. The system does not represent a black box approach; rather it has been integrated into the method currently used by the reporting officers at the Forensic Science Service (FSS). The time saved in automating routine calculations associated with mixtures analysis is significant. In addition, the computer program assists in unifying reporting processes, thereby improving the consistency of reporting.     

Interpreting simple STR mixtures using allele peak areas

Although existing statistical models can interpret mixtures qualitatively based upon the alleles present, the use of automated sequencers opens the opportunity to take account of quantitative aspects embodied by the peak area. One step in understanding simple mixtures consisting of just two donors is to estimate the mixture ratio. This is relatively easy to do when four-allele mixtures are evident at a given locus. However, if the mixture consists of three or fewer alleles, the process it is not straightforward. We demonstrate that mixture estimates are consistent across all loci in a multiplex system. Once the mixture ratio is known, then the expected peak areas for any given combination of alleles can be estimated using a simple spreadsheet analysis.     

A MCMC method for resolving two person mixtures

In this paper a Monte Carlo Markov Chain (MCMC) method for resolving DNA mixtures containing at most four peaks per locus into a major and a minor contributor is presented. Unlike previous methods, this method can provide posterior probability assessments of the most probable genotype and a likely range for the mixing proportion. The proposed method is applied to two DNA mixtures where the true genotypes of the contributors are known. The method provides posterior probabilities of the genotypes of the contributes which concord strongly with the known facts.     

Empirical analysis of the STR profiles resulting from conceptual mixtures

Samples containing DNA from two or more individuals can be difficult to interpret. Even ascertaining the number of contributors can be challenging and associated uncertainties can have dramatic effects on the interpretation of testing results. Using an FBI genotypes dataset, containing complete genotype information from the 13 Combined DNA Index System (CODIS) loci for 959 individuals, all possible mixtures of three individuals were exhaustively and empirically computed. Allele sharing between pairs of individuals in the original dataset, a randomized dataset and datasets of generated cousins and siblings was evaluated as were the number of loci that were necessary to reliably deduce the number of contributors present in simulated mixtures of four or less contributors. The relatively small number of alleles detectable at most CODIS loci and the fact that some alleles are likely to be shared between individuals within a population can make the maximum number of different alleles observed at any tested loci an unreliable indicator of the maximum number of contributors to a mixed DNA sample. This analysis does not use other data available from the electropherograms (such as peak height or peak area) to estimate the number of contributors to each mixture. As a result, the study represents a worst case analysis of mixture characterization. Within this dataset, approximately 3% of three-person mixtures would be mischaracterized as two-person mixtures and more than 70% of four-person mixtures would be mischaracterized as two- or three-person mixtures using only the maximum number of alleles observed at any tested locus.     

Identification and separation of DNA mixtures using peak area information

We introduce a new methodology, based upon probabilistic expert systems, for analysing forensic identification problems involving DNA mixture traces using quantitative peak area information. Peak area is modelled with conditional Gaussian distributions. The expert system can be used for ascertaining whether individuals, whose profiles have been measured, have contributed to the mixture. It can also be used to predict DNA profiles of unknown contributors by separating the mixture into its individual components. The potential of our probabilistic methodology is illustrated on case data examples and compared with alternative approaches. The advantages are that identification and separation issues can be handled in a unified way within a single probabilistic model and the uncertainty associated with the analysis is quantified. Further work, required to bring the methodology to a point where it could be applied to the routine analysis of casework, is discussed.     

DIP-STR在不平衡混合DNA样本中的分析研究

当前,DNA检验技术作为打击犯罪的利器,在法医鉴定中发挥着巨大作用。但对于性侵、暴力犯罪等案件中提取的混合DNA样本,尤其是从受害人或嫌疑人的接触物上采集的高度不平衡混合DNA样本,利用常染色体STR检验方法得到的结果通常不是很理想。由于PCR扩增偏倚,从混合样本中检测出痕量DNA分型是一个巨大的挑战,也是当前法医DNA检验的一个难点。近年来的研究显示,利用新型连锁遗传标记DIP-STR,即结合缺失或插入多态性片段DIP(deletion–insertion polymorphisms)和STR的连锁位点,可以用来检测出混合DNA样本中任一性别和细胞起源的微量DNA,甚至在DNA混合比例高达1:1000时,DIP-STR标记的灵敏度、特异性仍旧相对较为理想。因此,DIP-STR标记的分析可以作为常染色体STR检验的有效补充。本文将对DIP-STR在不平衡混合DNA样本分析中的研究背景、方法及其应用前景进行综述。     

Natural DNA mixtures generated in fraternal twins in utero

Analysis of multiple genetic loci using short tandem repeats (STR) is widely used in human identity testing because the extensive polymorphism at these loci allows for a high degree of discrimination among individuals. We recently received a forensic case that included several pieces of evidence and reference blood samples. Upon initial testing, one of the suspects had a DNA profile that included three alleles at four of the nine loci tested (vWA, FGA, TH01, and D5S818). At each locus, two of the alleles appeared to be "major" alleles with a third "minor" allele present. The profile appeared to be a mixture of two people. Contamination of this first reference sample was suspected and a second, unopened blood specimen was requested from this individual. The DNA profile from this second reference specimen was identical to that of the original specimen at each locus. One of the evidence samples also displayed an identical mixed DNA profile matching that of the reference specimens mentioned above. The relative peak heights of the two "major" and one "minor" allele remained constant in all three samples. Additional background information revealed that the suspect had not received a bone marrow transplant or blood transfusion. However, it was disclosed that this individual is a fraternal (dizygotic) twin. We hypothesize that an exchange of blood cells between the fetuses occurred in utero and that the additional alleles present in these reference samples are derived from cells contributed by his twin sibling. No additional specimens from the suspect or his twin could be obtained for confirmation, and our hypothesis remains untested. Forensic scientists should be aware of this possibility when faced with a DNA profile in which extra alleles at multiple loci are detected.     

A comparison of smokeless powders and mixtures by capillary zone electrophoresis

The analysis of inorganic ions present in smokeless and muzzleloading powders has been performed using capillary zone electrophoresis (CZE). Previous publications have examined inorganic low explosives using CZE, but have not looked at the ion profiles from smokeless powders. In this report, seven commercially available smokeless powders were analyzed as unburned powder and burned residue. The results demonstrate that ionic profiles can be used to characterize smokeless powders. Our analysis also included a smokeless powder/ Pyrodex combination to determine if smokeless powder ions are distinguishable in a mixture; however, the high concentration of ions present in Pyrodex RS prevented its detection. In addition, five different smokeless powder samples as well as Pyrodex RS were collected for analysis subsequent to deflagration in fifteen plastic pipe bombs. The relative ion concentrations between these powders can be used to illustrate the differences between open burning and pipe bomb deflagration.     

二组分混合DNA样品STR图谱解释

对混合样品STR图谱的结果进行解释。实验模拟二组分DNA混合样品 ,复合扩增荧光检测 10个基因座 ,比较混合样品谱带 ,计算等位基因峰面积比。结果发现 :二组分DNA混合样品的等位基因数增加 ,样品的混合比例不同就出现峰面积的不平衡。在等位基因峰面积比值与样品组分混合比例接近时 ,可由峰面积比值推断混合样品的混合比例。在混合比例为 1∶2 0时 ,基本上检测不到来自少量混合成分的等位基因 ,表现为单一组分图谱 ;在混合比例为 1∶10时 ,含量低的组分的等位基因峰面积接近与主要组分的“Stutter”峰面积 ,与来自主要组分的等位基因峰面积差异很明显。能检出混合样品中少量成分等位基因的最高混合比例为 1∶10     

Probabilistic expert systems for handling artifacts in complex DNA mixtures

This paper presents a coherent probabilistic framework for taking account of allelic dropout, stutter bands and silent alleles when interpreting STR DNA profiles from a mixture sample using peak size information arising from a PCR analysis. This information can be exploited for evaluating the evidential strength for a hypothesis that DNA from a particular person is present in the mixture. It extends an earlier Bayesian network approach that ignored such artifacts. We illustrate the use of the extended network on a published casework example.     

Using FastID to analyze complex SNP mixtures from indoor dust

Forensically relevant single nucleotide polymorphisms (SNPs) can provide valuable supplemental information to short tandem repeats (STRs) for investigative leads, and genotyping can now be streamlined using massively parallel sequencing (MPS). Dust is an attractive evidence source, as it accumulates on undisturbed surfaces, often is overlooked by perpetrators, and contains sufficient human DNA for analysis. To assess whether SNPs genotyped from indoor dust using MPS could be used to detect known household occupants, 13 households were recruited and provided buccal samples from each occupant and dust from five predefined indoor locations. Thermo Fisher Scientific Precision ID Identity and Ancestry Panels were utilized for SNP genotyping, and sequencing was completed using Illumina^® chemistry. FastID, a software developed to permit mixture analysis and identity searching, was used to assess whether known occupants could be detected from associated household dust samples. A modified “subtraction” method was also used in FastID to estimate the percentage of alleles in each dust sample contributed by known and unknown occupants. On average, 72% of autosomal SNPs were recovered from dust samples. When using FastID, (a) 93% of known occupants were detected in at least one indoor dust sample and could not be excluded as contributors to the mixture, and (b) non-contributor alleles were detected in 54% of dust samples (29 ± 11 alleles per dust sample). Overall, this study highlights the potential of analyzing human DNA present in indoor dust to detect known household occupants, which could be valuable for investigative leads.     

A method to enable forensic genetic genealogy investigations from DNA mixtures

The presence of more than one DNA contributor in an evidentiary sample may preclude attempts to use forensic genetic genealogy to develop an investigative lead. To address this issue, we developed a workflow for deconvolution of SNP mixtures into single source profiles that are suitable for matching against a genealogical database. Using the method, two-contributor DNA mixtures assayed using a commercial SNP typing kit can produce informative match results for both major and minor contributors.