Technical note: Development of DNA quantitation and STR typing systems for Panthera tigris species determination and individual identification in forensic casework

The aim of this technical note is to provide an overview of methodical approaches used to develop molecular systems for species determination/DNA quantification called Ptig Qplex and individual identification called Ptig STRplex of Panthera tigris samples. Both systems will help to combat the illegal trade of endangered species and create a worldwide shared database of DNA profiles.

The assessment of the efficacy of STRs panels recommended by the ISAG for canine pedigrees analysis for forensic casework

Canine DNA is widely used in forensic investigations, particularly in dog attacks cases on humans. Nowadays, STR markers are employed worldwide in forensic laboratories to test human and animal genotypes. In the study we analysed the effectiveness of panel – 18 STR as previously recommended by ISAG and the same panel with three additional markers – 21 STR, which has been recommended by ISAG as the core panel for dog identification since 2016. We calculated the PD, PID for these sets of panels and estimated RMP based on the DNA profile obtained during an investigation of a woman bitten by a dog. The high combined CPD value for 18 and 21 STRs showed values close to 1.0. The CPID value for theses panels was 5.2 × 10−10 to 6.4 × 10−14. Statistical analysis estimated the random DNA match, in the case of the woman bitten by a dog, with a probability of 4.3×1019 and 2.8×1022, using 18 and 21 STR panels respectively, and that the canine DNA profile from the crime scene originated from the suspected dog and not from another random dog. Our results show that both STR panels can be used effectively for individual identification and forensic casework.

Haplogroup Distribution of 309 Thais from Admixed Populations across the Country by HVI and HVII Sanger-Type Sequencing

The mitochondrial DNA (mtDNA) control region sequences for the hypervariable regions I (HVI) and II (HVII) of 309 Thai citizens were investigated using Sanger-type sequencing to generate an mtDNA reference dataset for forensic casework, and the haplogroup distribution within geographically proximal Asian populations was analyzed. The population sample set contained 264 distinct haplotypes and showed high haplotype diversity, low matching probability, and high powers of discrimination, at 0.9985, 0.4744%, and 0.9953, respectively, compared with previous reports. Subhaplogroup F1a showed the highest frequency in the Thai population, similar to Southeast Asian populations. The haplotype frequencies in the northern, northeastern, and southern populations of Thailand illustrate the relevance of social, religious, and historical factors in the biogeographical origin of the admixed Thai population as a whole. The HVI and HVII reference datasets will be useful for forensic casework applications, with improved genetic information content and discriminatory power compared to currently available techniques.

The human intervertebral disc as a source of DNA for molecular identification

Genetic analyses such as STR-typing are routinely used for identification purposes in forensic casework. Although genotyping techniques only require a minimum amount of DNA to provide a genetic profile, DNA quality differs not only between but also within tissues during ongoing decomposition. Initiated by a recent case where, due to the constitution of the body, preferred tissue was not available or only resulted in a partial and not usable DNA profile, the analysis of intervertebral discs as a source of DNA was considered. As the analysis of this tissue resulted in a high quality DNA profile a further study was performed in which thirty intervertebral discs dissected from bodies in different stages of decay were analyzed. All samples yielded good quality DNA in quantities suitable for STR-based amplification with no or only low degradation indices, resulting in complete genetic profiles. These results demonstrate the robustness of human intervertebral disc tissue as a source of DNA for molecular identification purposes.

Influencing Factors on Postmortem Protein Degradation for PMI Estimation: A Systematic Review

The present review provides an overview of the current research status on the effects of influencing factors on postmortem protein degradation used to estimate the PMI (postmortem interval). Focus was set on characteristics of internal and external influencing factors and the respective susceptibility and/or robustness of protein degradation. A systematic literature search up to December 2020 was conducted on the effect of influencing factors investigated in the context of postmortem protein degradation in the tissues of animals and humans using the scientific databases PubMed and Google Scholar, as well as the reference lists of eligible articles. We identified ten studies investigating a total of seven different influencing factors in degrading tissues/organs (n = 7) of humans and animals using six different methodological approaches. Although comparison of study outcomes was impeded by the high variety of investigated factors, and by high risk of bias appraisals, it was evident that the majority of the influencing factors concerned affected protein degradation, thus being able to modulate the precision of protein degradation-based PMI estimation. The results clearly highlight the need for a thorough screening for corresponding factors to enable the introduction of appropriate correction factors and exclusion criteria. This seems especially relevant for the protein degradation-based study of human PMI to increase the reliability and precision of the method and to facilitate a broader applicability in routine forensic casework.

A Single Method for 127 Recommended and Additional DUID Drugs in Blood and Urine by LC-MS/MS

Driving under the influence of drugs (DUID) cases continue to challenge forensic toxicologists as both the volume and complexity of casework increases. Comprehensive DUID testing should also meet the drafted ASB/ANSI standard and the NSC-ADID recommendations. A simple method using protein precipitation followed by filtration extraction with an 8-minute run time by LC-MS/MS was developed, and a comprehensive ASB/ANSI validation performed. Assessed in blood quantitatively, and urine qualitatively, is 127 target drug and metabolite analytes including cannabinoids (12), amphetamines (11), cocaine and metabolites (6), benzodiazepines (36), Z-drugs (5), opioids (27), anticonvulsants (3), first-generation antihistamines (6), muscle relaxants (2), dissociatives and hallucinogens (6), barbiturates (10), and miscellaneous substances (3). Limits of detection are appropriate for DUID, and other forensic casework such as drug-facilitated crime (DFC) and postmortem investigations. To demonstrate applicability, 78 proficiency test blood and urine samples, and 1,645 blood and urine samples from authentic cases samples demonstrated effective detection of target analytes in forensic casework. By increasing the analytical scope of multiple drug classes via a single method, this technique detects drugs that may have previously gone undetected, such as flualprazolam, etizolam, mitragynine, gamma-hydroxybutyric acid, and psilocin, and improves laboratory efficiency by reducing the number of tests required. The described method is, to the authors’ best knowledge, the only published single procedure to meet all drugs listed in the drafted ASB/ANSI standard, and recommended Tier 1 and traditional drugs from Tier 2 for DUID screening, whilst also achieving many drugs recommended for DFC and postmortem testing.

Nanopore sequencing in non-human forensic genetics

The past decade has seen a rapid expansion of non-human forensic genetics coinciding with the development of 2nd and 3rd generation DNA sequencing technologies. Nanopore sequencing is one such technology that offers massively parallel sequencing at a fraction of the capital cost of other sequencing platforms. The application of nanopore sequencing to species identification has already been widely demonstrated in biomonitoring studies and has significant potential for non-human forensic casework, particularly in the area of wildlife forensics. This review examines nanopore sequencing technology and assesses its potential applications, advantages and drawbacks for use in non-human forensics, alongside other next-generation sequencing platforms and as a possible replacement to Sanger sequencing. We assess the specific challenges of sequence error rate and the standardisation of consensus sequence production, before discussing recent progress in the validation of nanopore sequencing for use in forensic casework. We conclude that nanopore sequencing may be able to play a considerable role in the future of non-human forensic genetics, especially for applications to wildlife law enforcement within emerging forensic laboratories.