DEPArray™ single-cell technology: A validation study for forensic applications.

In forensics investigations, it is common to encounter biological mixtures consisting of homogeneous or heterogeneous components from multiple individuals and with different genetic contributions. One promising mixture deconvolution strategy is the DEPArray™ technology, which enables the separation of cell populations before genetic analysis. While technological advances are fundamental, their reliable validation is crucial for successful implementation and use for casework. Thus, this study aimed to 1) systematically validate the DEPArray™ system concerning specificity, sensitivity, repeatability, and contamination occurrences for blood, epithelial, and sperm cells, and 2) evaluate its potential for single-cell analysis in the field of forensic science. Our findings confirmed the effective identification of different cell types and the correct assignment of successfully genotyped single cells to their respective donor(s). Using the NGM Detect™ Amplification Kit, the average profile completeness for diploid cells was approximately 80%, with ∼ 290 RFUs. In contrast, haploid sperm analysis yielded an average completeness of 51% referring to the haploid reference profile, accompanied by mean peak heights of ∼ 176 RFUs. Although certain alleles of heterozygous loci in diploid cells showed strong imbalances, the overall peak balances yielded acceptable values above ≥ 60% with a mean value of 72% ± 0.21, a median of 77%, but with a maximum imbalance of 9% between heterozygous peaks. Locus dropouts were considered stochastic events, exhibiting variations among donors and cell types, with a notable failure incidence observed for TH01. Within the wet-lab experimentation with >500 single cells for the validation, profiling was performed using the consensus approach, where profiles were selected randomly from all data to better mirror real casework results. Nevertheless, complete profiles could be achieved with as few as three diploid cells, while the average success rate increased to 100% when using profiles of 6-10 cells. For sperms, however, a consensus profile with completeness >90% of the autosomal diploid genotype could be attained using ≥15 cells. In addition, the robustness of the consensus approach was evaluated in the absence of the respective reference profile without severe deterioration. Here, increased stutter peaks (≥ 15%) were found as the main artifact in single-cell profiles, while contamination and drop-ins were ascertained as rare events. Lastly, the technique's potential and limitations are discussed, and practical guidance is provided, particularly valuable for cold cases, multiple perpetrator rapes, and analyses of homogeneous mixed evidence.

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis.

Touch DNA has become increasingly important evidence in todays' forensic casework. However, due to its invisible nature and typically minute amounts of DNA, the collection of biological material from touched objects remains a particular challenge that underscores the importance of the best collection methods for maximum recovery efficiency. So far, swabs moistened with water are often utilized in forensic crime scene investigations for touch DNA sampling, even though an aqueous solution provokes osmosis, endangering the cell's integrity. The aim of the research presented here was to systematically determine whether DNA recovery from touched glass items can be significantly increased by varying swabbing solutions and volumes compared with water-moistened swabs and dry swabbing. A second objective was to investigate the possible effects of storage of swab solutions prior to genetic analysis on DNA yield and profile quality when stored for 3 and 12 months, as is often the case with crime scene samples. Overall, the results indicate that adapting volumes of the sampling solutions had no significant effect on DNA yield, while the detergent-based solutions performed better than water and dry removal, with the SDS reagent yielding statistically significant results. Further, stored samples showed an increase in degradation indices for all solutions tested, but no deterioration in DNA content and profile quality, allowing for unrestricted processing of touch DNA samples stored for at least 12 months. One further finding was a strong intraindividual change in DNA amounts observed over the 23 deposition days which may be related to the donor's menstrual cycle.

The DNA-Buster: The evaluation of an alternative DNA recovery approach.

Touch DNA recovery techniques can have limitations, as their effectiveness depends on the substrate on which the DNA of a person of interest can be found. In this study, an in-house dry-vacuuming device, the DNA-Buster, was compared to traditional methods for its DNA recovery performance from items typically examined in forensic casework. The aim was to evaluate whether this dry-vacuuming approach can recover DNA efficiently, potentially complementing the well-established recovery strategies. For this, the performances of swabbing, taping, wet- (M-Vac®) and dry-vacuuming (DNA-Buster) were investigated quantitatively and qualitatively for touch DNA deposited on carpet, cotton sweater, stone, tile and wood. For the sweater, both vacuuming methods outperformed the other collection tools quantitatively. While the highest DNA amounts for the carpet were yielded by swabbing and taping, dry-vacuuming was equally good in reaching full DNA profiles, whereas less complete profiles were observed for the M-Vac®. For stone and tile, swabbing was optimal, whereas dry-vacuuming clearly underperformed for these substrates. Taping was the best recovery method for wood. Despite applying single donor DNA after thoroughly cleaning the items, undesired DNA mixtures were detected for all recovery techniques and all substrates. The overall research findings show first that the novel dry-vacuuming method is suited for DNA recovery from textiles. Secondly, they indicate that more attention should be paid to the substrate-collection dependency to ensure best practices in recovering genetic material in a precise, confident and targeted manner from the variety of forensic casework material.

Collaborative swab performance comparison and the impact of sampling solution volumes on DNA recovery.

The collection of DNA traces marks the first step determining the success of genetic analysis. This study aimed to identify and validate a suitable alternative to the currently used ForensiX Evidence Collection Kit containing a cardboard box for swab storage. This box has to be folded at the crime scene, which is time-consuming and carries the risk of potential contamination and handling difficulties. A collaboration study involving three police departments and one laboratory for forensic genetics was performed to compare the currently used swab against three challenger swabs: ForensiX SafeDry, Copan 4N6FLOQSwab™ Genetics and Copan 4N6FLOQSwab™ Crime Scene. Mock samples consisted mainly of touch DNA, but also blood, saliva and semen were applied to twelve items with different surfaces. Every organisation contributed with three DNA collectors, whose individual collection efficiencies were investigated. The challenge of preparing homogenous traces, especially touch DNA, was addressed by enhancing hand contact frequency and sampling area. As a further part of the swab comparison study, we describe for the first time the influence of different swabbing solution volumes on the sampling efficiency of the different swabs. The application of touch DNA was also tested for a further swab type, the Sarstedt Forensic Swab, which yielded such low DNA concentrations that it was excluded from the collaboration study. The Copan Genetics and Copan Crime Scene swabs yielded significantly lower DNA concentrations than the currently used ForensiX Evidence Collection Kit and ForensiX SafeDry swab. The inter-individual performance results of the operators revealed significant differences in sampling skills. Comparing different swabbing solution volumes showed higher DNA yields or no significant difference for the ForensiX Evidence Collection Kit and ForensiX SafeDry than the Copan Genetics, depending on the item or trace type swabbed. Our results highlight the importance of validating first-step components that are decisive to the success of DNA typing in the context of specific sampling procedures and laboratory methods. Also, the significance of individuals' securing variations, principally unknown for crime scene investigation and laboratory teams, is emphasised for the first time, offering a practical approach for improving and training DNA collecting activities and ensuring the optimal securing evidence process. These findings increase the knowledge of impacts on DNA collection and, thus, benefit other laboratories and forensic services, particularly when using the same extraction methods.