In 2018, the Oregon State Medical Examiner’s Office (OSME) received a grant from the Bureau of Justice Assistance to use investigative genetic genealogy (IGG) with Parabon NanoLabs (Parabon) to attempt to make identifications in their unidentified human remains cases. Samples were screened for DNA quantity and quality, and selected samples were sent for genome-wide single nucleotide polymorphism (SNP) generation. This multi-year collaboration has been extraordinarily successful: of 43 cases submitted, 39 cases have been investigated using IGG, and 30 have resulted in an identification so far.
Frequency estimation for Y-STR haplotypes is a challenging problem because limited data are available and complex dependencies exist within the data. As a result, various statistical methods have been proposed for frequency estimation. The discrete Laplace method has been recommended in some contexts by the DNA commission of the ISFG. This method is limited to haplotypes with single integer repeat alleles only at all loci. We propose a generalisation of the method that handles duplicated loci such as DYS385 and less common alleles that are not integer repeats. The extension is implemented in an experimental R package called disclapmix2.
SE33 was a well-known autosomal short tandem repeat (STR) marker that was high polymorphic and therefore was high discrimination power. The sequence structure of STR markers has been increasingly explored with nextgeneration sequencing (NGS) technology. The sequencing resulted in the development of a new locus designation and allele nomenclature that was also backward compatible with the conventional capillary electrophoresis. SE33 was one of the STR markers that had been coamplified by ForenseqTM Signature Prep Kit (Verogen) but were not analyzed and illustrated in the Universal Analysis Software (UAS) (Verogen). This study reported an ambiguous sequence-based allele 16.3 of the SE33 locus. This allele was observed while analyzed by STRait Razor 3.0. The configuration file was modified from the previous studies to include 15 bp of 5 & PRIME; flanking region and 24 bp of 3 & PRIME; flanking region. The ambiguous allele was called 16.3 (106 bp) with a read count of 2070. However, the sequence of the repeat region cannot be designated as allele 16.3. Several possible scenarios for allele designation were presented and discussed.
Cannabis plants have been cultivated around the world for thousands of years and are widely used in the textile industry and medicine. From the point of view of analyses for the needs of the justice system, they can be divided into two categories - hemp of the fibrous type and the narcotic type, which differ in the content of the psy-choactive compound THC. According to the guidelines, varieties with a content of active compounds above 0.2 % are considered to be the narcotic type. The standard approach used to distinguish between these two types of Cannabis is commonly used toxicological analysis. However, if the content of differentiating active compounds or the type of plant part sent to the laboratory is problematic, genetic identification may be a good solution. The subject of the study of the research consortium was the genetic analysis of Cannabis samples using STR markers. The collected population database of samples of Cannabis from fibrous and narcotic varieties was analyzed taking into account the basic statistical parameters describing the population. It was used to develop a method of genetic profiling of Cannabis on the basis of a selected set of 23 STR genetic markers, which were subjected to preliminary statistical analyses. The developed genetic models will be used to select the most useful genetic markers for the genetic identification of Cannabis and will enable the creation of an identification tool for forensic experts.
The evaluation of forensic DNA expert opinions (in some countries expert witness testimonies) and the way it affects criminal judgement is of paramount importance. We have selected one of the largest challenges when it comes to the evaluation of forensic DNA evidence, contamination of DNA samples, and examined how it influences the decisions judges make about the credibility of DNA evidence in Hungary.
Assigning NoC in a mixed STR profile is an important preliminary step in computing a likelihood ratio (LR). A common metric is maximum allele count (MAC) whereby the locus exhibiting the largest number of alleles is used to set the NOC. This metric can be supplemented by considering total allele count (TAC) and locus allele count (LAC). TAC is the total number of alleles across all loci and is compared with probability distributions generated in silico. LAC works similarly, save that the probability distributions are generated at the locus level. Herein, we present a comparative analysis of these three metrics using a dataset of 10,000 of each of 2–7 person simulated ground truth mixtures. These datasets were used to generate parameter distributions for each NoC. This analysis showed LAC to be the most accurate single metric in all circumstances tested. We have developmentally validated an excel-based tool to automate calculations for use by operational caseworkers.
The EZ1&2 DNA Investigator Kit allows automated purification of total DNA from forensic and human identity samples on the EZ2 Connect Fx instrument. Its proven magnetic-bead technology provides high-quality DNA, suitable for direct use in sensitive downstream applications such as quantitative PCR, STR analysis and NGS. The purified DNA obtained is free of proteins, nucleases and inhibitors. In this study, the performance of the EZ1&2 DNA Investigator Kit on the EZ2 Connect Fx was evaluated using typical casework sample types and conditions commonly encountered in forensic and parentage laboratories. Wherever applicable, this validation study followed the recommendations of the European Network of Forensic Science Institutes (ENFSI) and the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM).
In this study, Japanese and Han Chinese individuals (n = 32, each) were genotyped for 261 autosomal STRs, and allele frequencies were calculated for each locus in each population. The average number of alleles for all loci in Japanese and Han Chinese populations was 6.65 and 6.56, respectively. The tests for deviations from HWE performed using an exact test showed that the number of STRs (P > 0.05) in Japanese and Han Chinese populations was 236 and 241, respectively. Calculation of forensic parameters showed heterozygosity, and the exclusion means in the Japanese population were 0.7185 and 4813 and those in the Han Chinese population were 0.7308 and 0.5008. In addition, population genetic analyses, such as principal component analysis and factorial correspondence analysis, were performed and a differential formula with likelihood ratios was applied for various number of STR loci based on the effectiveness of differentiation between the two populations. Accordingly, this study suggests that statistical differentiation between genetically close populations, such as the Japanese and Han Chinese populations, is possible if approximately 40-50 effective STR loci are analyzed.
In order to prioritize the exhumation of the most informative reference relatives to increase the statistical power of a reference group, a conditional simulation approach for missing person identification that combines both exclusion and inclusion power in reference families has been previously developed. The aim of this study is to empirically validate this approach by comparing its predicted theoretical prioritization model with the observed changes in statistical power in real cases of our laboratory, in which new relatives had already been added. We conclude that this approach is a reliable tool to choose the most appropriate reference relatives to complete a family group and improve the identification power of a Missing Person (MP).
The transfer, persistence, prevalence, and recovery of DNA (DNA-TPPR) can be highly relevant in forensic investigations to evaluate the presence and/or actions of a person of interest (POI). Whilst the DNA-TPPR-related research has increased significantly over the last decade, there is a lack of data on companion animals and their relationship to human DNA transfer. Given the commonality of cats and dogs in households around the world, companion animals as receptors and vectors for DNA transfer can be highly relevant in cases involving animals as victims of a criminal offense, or cases requiring activity level evaluations. Samples were collected from an external area on the right side of 20 cats to determine the prevalence and sources of human DNA on this area. Preliminary data shows that human DNA is present on household cats, its source is mainly from household inhabitants. Further studies are required to elucidate the means and level of transfer of human DNA to and from cats and other household animals. This knowledge can be relevant to sample targeting in specific case circumstances and/or when considering possible means of the presence of a person's DNA at the crime scene location.
Cow, Bos taurus, and female buffalo, Bubalus bubalis, are considered sacred animals that are a part of rural livelihood in India. The purity of products from these bovine species has significant sentimental implications in the dairy and meat industry. Therefore, the mitochondrial DNA and the sex origin, targeting the X and Y chromosomes from these bovine species, were selected to design three multiplex real-time probe PCR assays: HiPCR (R) Cow Detection Kit (MBPCR184), Hi-PCR (R) Buffalo Detection Kit (MBPCR185) and Hi-PCR (R) Cattle Sex Determination Kit (MBPCR186). Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines were followed to perform different studies using reference control DNAs. An Internal Reagent Control (IRC) was part of every assay, thus ensuring a successful reaction. The assays were 100% specific, with no crossamplification of the two bovine species. The amplification of the X chromosomal target was observed for male and female DNAs, whereas Y chromosome amplification was observed only for the male DNA. The assays were 100% specific to the target genes in these organisms with no non-specificity towards any other targets or organisms. The limit of detection for sex determination was 0.01 ng/mu l, whereas the differential capability of the assay was 3 copies/mu l and 30 copies/mu l for Bos taurus and Bubalus bubalis, respectively. The assays were reproducible at 1 ng/mu l genomic DNA with 95% CI. The assays are open and compatible with other brands of RealTime PCR systems used in forensic labs. The experiments presented here verify that the developed real-time PCR assays are robust, produce reliable and reproducible results for detection and differentiation of Bos taurus and Bubalus bubalis and their sex even at low DNA concentrations.
Large quantities of human cells and DNA fragments are continually released into the air and this constitutes a significant proportion of indoor dust. This study explored the feasibility of aerosol DNA as a source of infor-mation to aid human identification in criminal cases. Air samples were collected using the AirPrepTM ACD220 electret filter air sampler from indoor environments. Dust samples were collected in parallel from surfaces in the same areas. A new method to extract human DNA from the air filters was developed. DNA profiles were eval-uated in relation to the use and time of occupancy of the room from where samples were collected. This study showed that human DNA can be collected from air in sufficient amounts to yield full STR genotypes, suggesting the potential use of aerosol DNA as a novel investigative tool for forensic applications.
We analyzed the accumulation of population polymorphism in 2504 individuals -nuclear genomes (nDNA) of 26 populations (81 genes associated to extreme environments) and 3295 mitochondrial genomes (mtDNA) of 47 populations with the aim to found mitonuclear relationship associated an extremes environment as altitude.For that, we use an algorithm developed by us to determine the accumulation of polymorphisms by segments in the genome and thus be able to perform the multivariate analysis to found SNPs differences and similarities among populations.The results showed in Peruvian population a statistically significant mitonuclear relationship for 113/293970 nDNA SNPs in 16/81 genes.In the case of the mtDNA, we found a statistically significant mitonuclear relationship for 6/22 mtDNA positions -Gene.Additionally for the Peruvian population, the MRPP3 had the greatest polymorphism contribution with respect to other populations.Then, these nDNA and mtDNA SNPs in genetically close populations to Peru can be applied to forensic genomic phenotyping to identify groups likely adapted to extreme conditions (such as altitude) or make individualization between low and high altitude populations.
MicroHapulator’s empirical microhaplotype calling algorithm produces profiles well-suited for forensic analyst interpretation and probabilistic interpretation.
Latent DNA detection has the potential to transform aspects of DNA collection at scenes and from items. In the absence of being able to visualise the location of cellular material, all collection of samples at crime scenes is currently performed blind. With the advent of the application of a nucleic acid staining dye, the DNA within skin cells (commonly called keratinocytes and corneocytes) can be visualised. Diamond Dye fluoresces when it binds to the backbone of DNA. This fluorescence can be recorded using a simple mini-microscope allowing the location and number of cells to be recorded. The potential to visualise cells on a wide range of substrates opens the possibility to target sample collection and to triage samples for further analyses to only those containing DNA. Diamond Dye has been found to be safe at the concentration used, inexpensive, available commercially, easy to apply, is highly sensitive, and does not inhibit further analyses such as PCR. This work presented at the ISFG congress gives an overview of the current developments on using DNA staining dyes to record the number of cells present on a wide range of substrates. It is essential to firstly understand the composition of cellular material deposited by touch, where it originates and the relative composition of corneocytes and cell-free DNA. Insight into the origins of touch DNA will be presented along with the staining of nuclei using a range of dyes to show corneocyte degradation. The presentation will cover how DNA binding dyes can be used to effectively triage sample collection, monitor cell collection using different swabs and tapes.
Forensic genetic laboratories are challenged with implementing innovation even if the benefits to operational performance are well demonstrated often because of internal budget constraints. A prospective cost–benefit analysis (CBA) could support justification for an increased budget by effectively demonstrating in a system-based approach the relatively small cost of increasing a laboratory budget can substantially reduce costs to society (both qualitatively and monetarily). A Monte Carlo simulation and sensitivity CBA was performed using a more expensive swab (i.e., nylon 4N6FLOQSwabs®) compared with a less costly cotton swab. Ranges of input values and tangible and intangible benefits were considered. The outcome is that the relatively small increased cost of using a nylon swab pales compared with the potential tangible and intangible benefits to the overall system. This approach provides a sounder basis for requesting additional funds to support implementation of technologies and better approximates realistic situations while accommodating uncertainty of input values.
The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.
Biological samples recovered from recipients of allogeneic haematopoietic stem cell transplant (HSCT) contain genetic material from both donor and him/herself. This chimeric condition can greatly complicate analysis of a DNA evidence and undermine its power of discrimination, as the specimens could be mistakenly identified as a mixed sample when in fact it originated from a single person. Profiling reference samples could help clarify the profile anomalies, however, the degree of mixture between host's and donor's genetic materials has been reported to vary depending on the tissue from which DNA were obtained. As a result, knowledge to select appropriate sources of reference samples that would most likely reveal the person's pre- and post-transplant alleles becomes necessary to minimise any possible misunderstanding during the subsequent DNA profile interpretation and comparison. This work investigated the extent of chimerism present in different types of biological samples collected from an individual who had undergone a bone marrow transplant as a child (posttransplant interval, PTI >28 years). DNA profiles from buccal cells, saliva, hair roots, and fingernail clippings were generated using AmpFlSTRTM IdentifilerTM Plus/Direct PCR Amplification Kit. The donor's STR profile was used to identify donor- and host-specific alleles (DSA and HSA, respectively), and relative donor chimerism (% Ch) of each marker was calculated. Results showed that the saliva sample contained the highest level of chimerism, with complete profile of donor being detected and the mean %Ch was 34.0 & PLUSMN; 4.5. Four out of eight DSA dropped out from the buccal cell profile, and the mean %Ch was 24.2 & PLUSMN; 1.7. Hair roots and fingernail clippings yielded single source profiles and only host's original alleles were present. Except for the fingernail results, these observations agreed with many of the previous studies, which further demonstrated the need for raising awareness among forensic genetic laboratories regarding the type of reference samples that should be collected in future cases.
The present study aimed to characterize the paternal genetic composition of the Ecuadorian indigenous population. To investigate a possible matting bias, AIM-InDels were also analyzed. The population sample of 54 self-declared Ecuadorian indigenous presented a high haplotype diversity. A major proportion of the male lineages belong to Native American haplogroups. Although from self-declared indigenous, the studied population sample presented a low genetic distance with a sample of admixed individuals from Ecuador.
After developing an LR-system to calculate weight of evidence from mRNA data [1] we here present how this LR-system is used in forensic casework. We aim to convey the weight of the evidence in forensic reports.
