Validating TrueAllele® DNA mixture interpretation

Abstract: DNA mixtures with two or more contributors are a prevalent form of biological evidence. Mixture interpretation is complicated by the possibility of different genotype combinations that can explain the short tandem repeat (STR) data. Current human review simplifies this interpretation by applying thresholds to qualitatively treat STR data peaks as all‐or‐none events and assigning allele pairs equal likelihood. Computer review, however, can work instead with all the quantitative data to preserve more identification information. The present study examined the extent to which quantitative computer interpretation could elicit more identification information than human review from the same adjudicated two‐person mixture data. The base 10 logarithm of a DNA match statistic is a standard information measure that permits such a comparison. On eight mixtures having two unknown contributors, we found that quantitative computer interpretation gave an average information increase of 6.24 log units (min = 2.32, max = 10.49) over qualitative human review. On eight other mixtures with a known victim reference and one unknown contributor, quantitative interpretation averaged a 4.67 log factor increase (min = 1.00, max = 11.31) over qualitative review. This study provides a general treatment of DNA interpretation methods (including mixtures) that encompasses both quantitative and qualitative review. Validation methods are introduced that can assess the efficacy and reproducibility of any DNA interpretation method. An in‐depth case example highlights 10 reasons (at 10 different loci) why quantitative probability modeling preserves more identification information than qualitative threshold methods. The results validate TrueAllele^® DNA mixture interpretation and establish a significant information improvement over human review.     

mtDNA测序结果与Anderson标准序列的比对－ClustalX等共享软件在法医物证学上的应用

目的 对多个样本的线粒体DNA(mtDNA)高变区测序结果与Anderson标准序列进行比对分析。方法 利用ABI测序仪测定生物学样本的mtDNA高变区序列,得到测序结果文件,通过Chromas、 SeqVerter软件将之转换为aln文件,用ClustalX软件与Anderson标准序列(txt文件)进行比对,确定突变点的碱基排列次序和位置。结果Chromas、SeqVerter和ClustalX软件界面友好,操作简便,可以方便地用于多个样本DNA序列的比较,结果直观,易于判读。结论　运用Chromas、SeqVerter和ClustalX等共享软件,可成功地对多个样本的mtDNA高变区序列进行比对分析。     

The use of the LightCycler for the detection of Y chromosome SNPs

A novel methodology based on PCR monitoring on-line with fluorescent formats using the LightCycler for Y chromosome SNP typing is proposed. The main advantages of the system are the time necessary for the analysis (which is around 20 min), the robustness and the accuracy of the method and especially its sensitivity, which permits the detection of the male component in male-female mixtures up to 1:300 for some of the SNPs.Singleplexes of four different SNPs (M9, sY81, SRY-1532 and SRY-2627) as well as two duplexes (M9 and sY81 on the one hand and SRY-1532 and SRY-2627 on the other) were efficiently implemented. A simultaneous amplification and analysis of the four SNPs is also possible. It seems difficult with the current methodology to implement more than a quadruplex.     

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.     

DNA mixture genotyping by probabilistic computer interpretation of binomially-sampled laser captured cell populations: Combining quantitative data for greater identification information

Two person DNA admixtures are frequently encountered in criminal cases and their interpretation can be challenging, particularly if the amount of DNA contributed by both individuals is approximately equal. Due to an inevitable degree of uncertainty in the constituent genotypes, reduced statistical weight is given to the mixture evidence compared to that expected from the constituent single source contributors. The ultimate goal of mixture analysis, then, is to precisely discern the constituent genotypes and here we posit a novel strategy to accomplish this. We hypothesised that LCM-mediated isolation of multiple groups of cells (‘binomial sampling’) from the admixture would create separate cell sub-populations with differing constituent weight ratios. Furthermore we predicted that interpreting the resulting DNA profiling data by the quantitative computer-based TrueAllele® interpretation system would result in an efficient recovery of the constituent genotypes due to newfound abilities to compute a maximum LR from sub-samples with skewed weight ratios, and to jointly interpret all possible pairings of sub-samples using a joint likelihood function.As a proof of concept, 10 separate cell samplings of size 20 recovered by LCM from each of two 1:1 buccal cell mixtures were DNA-STR profiled using a specifically developed LCN methodology, with the data analyzed by the TrueAllele® Casework system. In accordance with the binomial sampling hypothesis, the sub-samples exhibited weight ratios that were well dispersed from the 50% center value (50 ± 35% at the 95% level). The maximum log(LR) information for a genotype inferred from a single 20 cell sample was 18.5 ban, with an average log(LR) information of 11.7 ban. Co-inferring genotypes using a joint likelihood function with two sub-samples essentially recovered the full genotype information. We demonstrate that a similar gain in genotype information can be obtained with standard (28-cycle) PCR conditions using the same joint interpretation methods. Finally, we discuss the implications of this work for routine forensic practice.     

mtDNA investigations after differential lysis

In order to gain information regarding the ethnic origin of an unknown offender in a murder case it was necessary to sequence the HV1 and 2 regions of the mitochondrial DNA (mtDNA). The only evidentiary material that could be linked to the suspect was DNA, extracted from spermatozoa after differential lysis. The observed mtDNA sequences were identical to the sequences of the victim. Therefore, we had to check if this was a coincidence or the result of a technical limitation of the testing procedure. Subsequently, we performed a systematic study. In cases with complete separation of sperm and female cells it wasn't possible to obtain a mtDNA sequence for the sperm fraction. This phenomena is based on the loss of the sperm's flagellum and mid-piece during the first lysis step and a concomitant loss of the sperm's mitochondria. In our murder case, a minor carry-over of female cells to the sperm fraction was responsible for the sequencing result.     

Quick Screening of Crystal Methamphetamine/Methyl Sulfone Exhibits by Raman Spectroscopy

Abstract: The analysis of mixtures of “crystal meth” (usually comprised of methyl sulfone [MS] and methamphetamine [MA]) by gas chromatography‐mass spectrometry (GCMS) is routine in many forensic drug laboratories. The utilization of Raman spectroscopy for the identification of such mixtures quickly and without the need for a separation technique is discussed. Samples were dissolved in water and Raman spectra of the resulting aqueous solutions were collected. By comparing these spectra to spectra of methylsulfone and MA mixtures of known composition, an indication of the composition of the sample can be obtained in only a few minutes. This spectral comparison also can be used as a semi‐quantitative analysis of MA concentrations in such exhibits.     

Probabilistic genotyping of single cell replicates from complex DNA mixtures recovers higher contributor LRs than standard analysis

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2–6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥10²⁰) from donors that had returned non-probative LRs (<10³) by standard methods.     

Comparative Analysis of the HV1 and HV2 Regions of Human Mitochondrial DNA by Denaturing High-Performance Liquid Chromatography

Abstract:  Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D-loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.     

The development of a filter model for prioritising suspects in burglary offences

Abstract

This paper discusses the development of a filter model for prioritizing possible links in dwelling burglary. The filters utilize the central aspects of crime scene information that is available and accessible to investigators in burglary, namely geo-spatial, temporal, behavioural, and dwelling information. The proposed filters were analysed using a sample of 215 dwelling burglaries committed by 43 serial burglars (i.e. 5 offences each) in order to determine the sequence in which the filters should be considered in prioritizing possible linked offences. The results indicated that the following order (i.e. better performance to worse performance) was most effective at linking offences, utilizing: (1) geo-spatial information, (2) temporal aspects, (3) behavioural information and, lastly, (4) dwelling characteristics. Specifically, the results indicated that offences in close proximity to one another should be given priority. Further, any offence occurring within a 28-day span before or after the index offence should be given priority. The paper argues that behavioural and dwelling characteristics are less effective for linking than geo-spatial and temporal information because the former two aspects are influenced significantly by situational and contextual cues on offender decision-making.     

Classification of black powder toners on the basis of integrated analytical information provided by Fourier transform infrared spectrometry and X-ray fluorescence spectrometry

Differentiating between black powder toners used in laser printers and copiers can be challenging for forensic examiners. One hundred and sixty-two samples from 82 different types of cartridges produced by 21 manufacturers that are currently available on the Polish market were studied using Fourier transform infrared spectrometry (FT-IR) and energy-dispersive X-ray fluorescence spectrometry (XRF). The spectra obtained by these two methods were classified into 14 groups and 28 groups using FT-IR and XRF, respectively. These classifications served as a basis for the development of an analytical scheme for differentiating black powder toners. This scheme can be used with any two methods that supply different information about an examined sample. The research will show that if two samples are similar in polymer composition (e.g., FT-IR spectra), additional quantitative elemental composition from XRF analyses may provide more discrimination. It was possible to differentiate 82.5% pairs of examined samples using only FT-IR, 90.8% pairs of examined samples using only XRF, and 95.8% pairs of examined samples using these two methods. Obtained spectra and all the available information could be used to create/build a database. The results obtained confirm the multiplicity and compatibility of toners. Additionally, it was stated that not all the samples were original (OEM).     

A comparison of forensic evidence recovery techniques for a windows mobile smart phone

Acquisition, decoding and presentation of information from mobile devices is complex and challenging. Device memory is usually integrated into the device, making isolation prior to recovery difficult. In addition, manufacturers have adopted a variety of file systems and formats complicating decoding and presentation.A variety of tools and methods have been developed (both commercially and in the open source community) to assist mobile forensics investigators. However, it is unclear to what extent these tools can present a complete view of the information held on a mobile device, or the extent the results produced by different tools are consistent.This paper investigates what information held on a Windows Mobile smart phone can be recovered using several different approaches to acquisition and decoding. The paper demonstrates that no one technique recovers all information of potential forensic interest from a Windows Mobile device; and that in some cases the information recovered is conflicting.     

Types of Theatre Information and Marketing-Audience Development Among the Alienated

Findings concerning audience development for the Performing Arts among the alienated are presented and discussed. The effects of different types of information upon attitude formation of the alienated toward the Performing Arts were investigated. The most effective type of information in forming positive attitudes toward the Performing Arts among the alienated was explaining how the individual can gain immediate benefits through supporting the theatre. Information that explained how the society would benefit in the future from support of the Performing Arts was much less effective in forming positive attitudes among the alienated. These findings are significant because the size of the potential audience among the alienated is large and growing, and because the alienated are in a position to be particularly able to benefit from the Performing Arts.     

电视节目模板的法律保护

电视节目模板已经在国际上形成了一个巨大的产业,但人们对它本身的认识及其法律保护问题仍存在着巨大的分歧。在各国现行的法律框架下,虽然电视节目模板的某些元素分别受到不同法律的保护,但电视节目模板作为一个整体往往不符合版权法的保护标准,也很难受到反不正当竞争法的保护。     

Hair analysis for opiates,cocaine and metabolites. Evaluation of a method by interlaboratory comparison

The aim of this study was to evaluate the performance of a technique for the simultaneous testing of opiates, cocaine and metabolites in hair by interlaboratory comparison. Sixteen forensic and clinical laboratories with different degrees of experience in hair analysis participated voluntarily in the study (no selection criteria were applied). The suggested analytical procedure, the one routinely used in our laboratory, consisted of incubation in HCl 0.1N (45 degrees C, overnight), solid phase extraction (with Bond Elut Certify) cartridges), derivatisation (trimethylsilyl (TMS) derivatives) and GC-MS analysis. Three different mixtures of finely cut (1 mm or less) hair were prepared using drug-users' and drug-free hair: one 'negative' sample (<0.1 ng/mg for morphine, 6-acetylmorphine (6AM), cocaine and benzoylecgonine (BE)), one 'low concentration' sample (between 0.5 and 2 ng/mg) and one 'high concentration' sample (>3 ng/mg). Accuracy and precision (CV% lower than 5.1, 9.9, 5.2, 3.8, 7.3 and 8.3% for morphine, 6AM, codeine, cocaine, BE, and methylecgonine (ME), respectively; range 0.5-5 ng/mg) of the method and homogeneity of the mixtures were evaluated in our laboratory by intraday (CV% lower than 12% for all analytes) and interday analyses (CV% lower than 17% for all analytes except 6AM, 25%). Participants in the study were grouped into: (1) laboratories (n = 6) obtaining the best qualitative and quantitative values, corresponding to those with long experience in hair analysis; (2) laboratories (n = 5) with no reported false positive and/or false negatives; (3) laboratories (n = 5) with one or more reported false positives/false negatives. The results obtained by the labs of the first group were used as reference values. The scatter of data was similar to those obtained in other published studies.     

An Evaluation of Standardized Software for Processing GC/MS Data from Different Vendors' Instruments in a Forensic Laboratory

Forensic science laboratories perform analyses on a variety of materials using gas chromatography/mass spectrometry (GC/MS). Instruments from different vendors may be used, requiring analysts to be proficient in the use of multiple proprietary software packages for collecting and processing data. There is no standardized GC/MS software available that can acquire data from different vendors' instruments. However, there are third‐party processing software products that can import data files in different formats. The Centre of Forensic Sciences compared the data processing performance of one such product, ACD/MS Manager Suite, with three instrument vendors' software used for casework analysis. This product was tested for its compatibility with the existing software, its capability to load and present data, and to initiate searches of commercial libraries. The study shows that the MS Manager module provides a means for the forensic analyst to view, process, and report on data from different sources in one software package.     

Introduction of the python script MHinNGS for analysis of microhaplotypes

MHinNGS is a Python application developed for analysis of microhaplotypes (MHs) in single-end sequencing data. MHinNGS analyses reads in standard formats and store each sequence into bins, one bin for each MH as defined by the two flanking sequences. MHinNGS requires a reference genome and a configuration file with information about each locus. Four mandatory and 15 optional criteria defined in the configuration file allow detailed locus-specific analyses of the MH loci. The program 1) removes noise, 2) identify and name alleles, 3) test the genotypes, and 4) test unique sequences not identified as noise or alleles. MHinNGS produces a result file, where every unique sequence that passed the noise filter is presented with MH allele, read depth, warning flags based on the genotyping criteria, sequence, heterozygote balance, and MH name. Furthermore, variation in other parts of the fragment that is not defined as SNPs in the MH, linked variants, or rare SNPs are listed in a separate column of the result file.     

Evidence on unusual way of cocaine smuggling: cocaine-polymethyl methacrylate (PMMA) solid solution--study of clandestine laboratory samples

An abandoned clandestine laboratory was seized in Slovenia. All confiscated exhibits were analysed in a forensic laboratory, where the following analytical methods were applied: capillary gas chromatography coupled with mass spectrometry (GC-MS) combined also by solid-phase micro extraction (SPME) and pyrolysis (Py) technique, Fourier transform infrared spectrometry (FTIR) and scanning electron microscopy with energy dispersive X-ray detector (SEM-EDX). The most interesting analytical findings can be summarised as follows: at the crime scene some plastic pieces, which contained cocaine dissolved (as solid solution) in polymethyl methacrylate-plexiglass (PMMA), were found. The highest cocaine concentration measured in the plastic sample was about 15% by weight. Two larger lumps of material (12 and 3 kg) were composed mainly of PMMA and CaCO3 and contained only 0.4 and 0.5% of cocaine, respectively. As for the low cocaine concentration, we assume that those two lumps of material represent discarded waste product--residue after the isolation of cocaine from plastic. Higher quantities of pure solvents (41 l) and solvent mixtures (87 l) were seized. We identified three types of pure solvents (acetone, gasoline and benzine) and two different types of solvent mixtures (benzine/acetone and gasoline/acetone). The total seized volume (87 l) of solvent mixtures holds approximately 395 g of solid residue formed mainly of PMMA and cocaine. Obviously solvent mixtures were used for isolation of cocaine from the plastic. Small quantities of relatively pure cocaine base were identified on different objects. There were two cotton sheets, most probably used for filtration. One sheet had traces of cocaine base (76% purity) on the surface, while cocaine in hydrochloride form (96%) was identified on the other sheet. GC-MS analyses of micro traces isolated from analytical balances showed the presence of cocaine and some common adulterants: phenacetine, lidocaine and procaine. A cocaine sample compressed in the shape of block was also seized. The above analytical findings inferred us to the conclusion that the illicit laboratory was used for the isolation of cocaine from PMMA resin. Further more, analyses confirm that not only isolation but also further manipulation of cocaine, i.e. adulteration/dilution, as well as the formation of cocaine blocks took place in the house. The information obtained through analyses also allowed us to make some hypotheses about possible multistage isolation procedure.     

Improving performance in a Swedish police traffic unit: Results of an intervention

This article describes the results of a feedback system designed to improve performance for a Swedish traffic police unit and examined whether such a feedback system was beneficial or detrimental to the attitudes of the officers. As in many Western countries, government organizations are being required to demonstrate their effectiveness with quantitative performance measures. An approach called the Productivity Measurement and Enhancement System (ProMES) was used with three groups of Swedish Traffic Police to do this. ProMES is a method for identifying unit's objectives, developing measures for these objectives, and using this information as feedback. ProMES was developed with these police units and feedback from the system was used over a four-year period. Results indicate that there were substantial increases in performance. There were also decreases in accidents, injuries, and fatalities compared both to baseline and to comparison groups in Sweden. These improvements were made with fewer and fewer police officers each year.     

Chemical development of latent fingerprints: 1,2-indanedione has come of age

The performance of 1,2-indanedione as a latent fingerprint reagent on some types of paper was found to exceed that of DFO, the leading fluorogenic fingerprint reagent. It even exceeds the performance of the sequence, DFO, followed by ninhydrin. No new prints could be observed when ninhydrin was applied after indanedione. On a large number of actual exhibits (used checks) indanedione developed 46% more identifiable prints than the sequence DFO-ninhydrin. A standard procedure for fingerprint development by indanedione is proposed. Best results are obtained with a 0.2% indanedione solution in HFE7100 solvent containing 7% ethyl acetate, but no acetic acid. It can be recommended to start using 1,2-indanedione, which is already commercially available, in actual fingerprint casework.