The identification of biological evidence is particularly important for criminal detection, and the deoxyribonucleic acid (DNA) analysis plays a significant role in reconstructing events. However, bloodstains after thermal exposure in fires are quite unique compared to those in other scenes. Previous results regarding DNA recovery in bloodstains after heating are inconsistent with each other, which limits guidance for forensic science. In order to confirm the influence of heat on DNA recovery, the important physical evidence, bloodstain, was selected for its common occurrence, and a standard heat source, the Cone Calorimeter, was used to simulate high temperatures in fire scenes. A series of bloodstains were prepared under different heating conditions, and the results of short tandem repeat (STR) typing were systematically correlated with the trace characteristics of bloodstains. The findings indicate that heating bloodstains below 150 °C for less than 10 mins has minimal effect on DNA testing. After heating bloodstains at 150 °C for 20 mins or at 180 °C for 5 mins, partial DNA profiles were obtained, accompanied by blackening and cracking of the bloodstains. After exposure to 180 °C for 20 mins or 200 °C for 10 mins, no DNA profiles were obtained with bloodstains exhibiting metallic lusters and black bulges. Furthermore, from the perspective of chemical bond energy, the C-N, C-O, C-C, and P-O bonds in DNA molecules are prone to breaking during heating. The C-N bond serves as the primary connection between the four bases and the strand, while the C-O, C-C, and P-O bonds are significant connections within the DNA strand. It is thus hypothesized that the breakage of any bond aforementioned during heating results in the failure of DNA typing. Understanding the correlation between trace characteristics of bloodstains and DNA typing results after thermal exposure is crucial for comprehending DNA recovery from physical evidence collected from fire scenes.

Sudden unexpected death (SUD) is an unexpected event that in many cases are caused by diseases with an underlying genetic background. Forensic molecular autopsy is an approach that has gained wide-spread attention, in part explained by the rapid progress of DNA sequencing techniques. The approach leverages genetic data in combination with medical autopsy findings in post-mortem samples to explore a potential underlying genetic cause of death. Traditional forensic approaches to molecular autopsy focus on a small panel of genes, say <200 genes, with strong association to heart conditions whereas clinical genetics tend to capture entire exomes while subsequently selecting targeted panels bioinformatically. The drop in price and the increased throughput has promoted wider exome sequencing as a viable method to discover genetic variants. We explore a targeted gene panel consisting of 2422 genes, selected based on their broad association to sudden unexplained death. A hybridization capture approach from Twist Bioscience based on double stranded DNA probes was used to target exons of the included genes. We selected and sequenced a total of 98 post-mortem samples from historical forensic autopsy cases where the cause of death could not be unambiguously determined based on medical findings and that had a previous negative molecular autopsy. In the current study, we focus on the performance of the hybridization capture technology on a 2422 gene panel and explore metrics related to sequencing success using a mid-end NextSeq 550 as well as a MiSeq FGx platform. With the latter we demonstrate that our sequence data benefits from 2×300 bp sequencing increasing coverage, in particular, for difficult regions where shadow coverage, i.e. regions outside the probes, are utilized. The results further illustrate a highly uniform capture across the panel of genes (mean fold80=1.5), in turn minimizing excessive sequencing costs to reach sufficient coverage, i.e. 20X. We outline a stepwise procedure to select genes associated with SUD through virtual bioinformatical panels extracting tier of genes with increasing strength of association to SUD. We propose some prioritization strategies to filter variants with highest potential and show that the number of high priority genetic variant requiring manual inspections is few (0-3 for all tiers of genes) when all filters are applied.

The work presented herein is the second part of a large-scale persistence project aimed at identifying trends in trace DNA persistence. This study aims to show how different environmental storage conditions and target surface characteristics influence the persistence of cellular and cell free DNA (cfDNA) over time. To eliminate variation within the experiment, we used a proxy DNA deposit consisting of a synthetic fingerprint solution, cellular DNA, and/or cfDNA. Samples were collected and analysed from eight non-metal surfaces over the course of 1 year (27 time points) under three different environmental storage conditions. The results of this experiment show that surface characteristics in conjunction with DNA type greatly influence DNA persistence. Variation in the amount of DNA recovered over time was greatly influenced by surface porosity. CfDNA persisted at significantly higher levels on non-porous surfaces, and cellular DNA persisted at higher levels on porous items. Furthermore, statistically significant differences in DNA persistence were found among the items classified as non-porous surfaces and among the items classified as porous surfaces. Additionally, this study showed that the sample storage environment had a larger impact on DNA persistence than previously observed for metal surfaces [1]. When considering DNA type, cellular DNA was shown to persist for longer than cfDNA and persistence as a whole appears to be better when DNA is deposited alone rather than in mixtures. Unsurprisingly, it was found that the amount of DNA recovered from trace deposits decreased over time. However, DNA decay is highly dependent on the surface type and exhibits higher variability at short time points and on porous surfaces. For each of the surfaces tested, DNA persisted 1 year past deposition (in some combination of DNA type and environmental condition), except for wood, on which DNA did not persist in any capacity past four months. This data is intended to add to our understanding of DNA persistence and the factors which affect it.

Biological traces recovered from crime scenes serve as vital evidence in forensic investigations. While DNA evidence is frequently used to address the sub-source level of the hierarchy of propositions, the biological source of the DNA can be highly probative at the source level. Current body fluid detection methods pose certain limitations, such as reports of false positive results from some of the presumptive and/or confirmatory tests in current use. These tests are also individual tests for the detection of one body fluid, meaning that if the sample is suspected to be a mixture of multiple body fluids, then different tests would need to be conducted to confirm the body fluid(s) present, which may exhaust small amounts of available biological trace. Proteomics applications for the identification of body fluids have been previously explored, and potential biomarkers indicative of body fluids discovered from liquid-chromatography tandem mass spectrometry (LC-MS/MS) methods have been reported. This work focuses on developing a mass spectrometry-based proteomics approach for the identification of body fluids by targeting discriminating peptide biomarkers from the non-DNA component left over after DNA extraction of samples. The non-DNA component is typically a waste product but with unappreciated evidential value. Our methodology for the purification of proteins from the post-DNA extraction waste includes an acetone precipitation and single-pot solid-phase-enhanced sample preparation (SP3) technique, microwave-assisted trypsin digestion, and LC-MS/MS analysis of the resultant peptides. Preliminary results from this proof-of-concept study include a list of potentially discriminating proteins and peptides for blood, saliva, and semen developed from the analysis of post-DNA extraction waste. Our method allows for multiple analytes to be targeted simultaneously from a DNA profiling waste stream and we anticipate that it could eventually be incorporated into standard forensic laboratory workflows.

This article presents a case where the issue was to determine who was the driver and who was the passenger at the time of a fatal car accident involving two persons, one of whom died in the accident. The presence of the two persons in the car was not contested, only the mechanisms that led to the deposition of the DNA (i.e., the activities) were. To our knowledge, few cases are evaluated considering the alleged activities. The reasons for this include the lack of knowledge, and data, as well as the difficulties encountered for the formulation of conclusions. In this case report, we present the architecture of the Bayesian Network (BN) used to evaluate the DNA results of the traces recovered from the steering wheel, driver's and passenger's airbags. The following propositions were considered: "The person of interest (POI) was driving the car and the alternative person (AP) was the passenger at the time of the accident" or vice versa. We discuss the assumptions that were made and how data from the literature was used to parametrize into the BN. A likelihood ratio of the order of 90 was finally assigned. The statement proposed to the mandating authority indicated that, given the information that was made available to us, our observations were of the order of 90 times more probable if the POI was driving the car at the time of the accident rather than if the AP was. A sensitivity analysis was performed (5000 simulations): this shows that our likelihood ratio is robust.

While often necessary in sexual assault cases, confirmatory identification of body fluids can be a lengthy and/or costly process. In particular, the detection of vaginal fluid and menstrual fluid in forensic casework is limited to endpoint reverse-transcription PCR to detect fluid-specific messenger RNA (mRNA) markers as there are no robust chemical or enzymatic techniques available for these fluids. Similarly, testing for rectal mucosa is not possible with standard methods, the presence of which would provide probative value in cases of alleged anal penetration, although mRNA-based markers have recently been described. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative technique that enables detection of mRNA at a single temperature (usually 60-65℃) for 10-30 minutes and has comparable sensitivity to PCR. We describe the coupling of RT-LAMP amplification (60℃ for 30 minutes) with CRISPR-mediated fluorescent detection of the body fluid specific mRNA markers MMP3 (menstrual fluid), CYP2B7P (vaginal material), TNP1 (spermatozoa), KLK2 (semen), and MUC12 (rectal mucosa). Following temperature optimization and final selection of RT-LAMP-CRISPR assays, their specificity across circulatory blood, buccal, menstrual fluid, vaginal material, semen, and rectal mucosa was assessed. Most assays were specific for their intended target body fluid, although MMP3 and CYP2B7P were detected in some rectal mucosa samples, the latter of which has been observed previously in the literature. A preliminary sensitivity assessment in target fluids was determined by a dilution series over six logs of RNA input. A range of assay approaches were investigated to develop a protocol suitable for use in a forensic screening laboratory. This included the determination of fluorescent assay results by eye, use of lyophilised reagents, and RT-LAMP and CRISPR reactions undertaken in one-tube in a lower resource setting.

Identity-informative single nucleotide polymorphisms (iiSNPs) are valuable genetic markers for human identification and kinship testing in forensic casework, especially when the quality and quantity of DNA evidence is not suitable for routine short tandem repeat (STR) profiling. This study analysed 105 buccal samples representing the Australian population with European ancestry in order to assign allele frequencies and conduct population genetic analyses for 94 iiSNPs and 20 STRs. The markers were assessed by calculating relevant forensic statistics and testing for deviations from Hardy-Weinberg and linkage equilibrium. No linkage of statistical significance was observed between any of the pair-wise combinations of the combined 114 identity-informative markers and only one STR exhibited deviation from Hardy-Weinberg equilibrium (D8S1179). The probability of matching genotypes being observed within this population was of the order of 10 for STRs, 10 for iiSNPs and 10 for the combined identity-informative marker panel, improving the ability to discriminate between individuals when calculating likelihood ratios in direct or indirect matching scenarios. Further, the addition of iiSNPs will facilitate identifications when suboptimal STR profiles are recovered from compromised or challenging samples and aid comparisons to genetic relatives for familial or kinship testing.

The microbiome is a promising tool for identifying body fluids which can be deposited on various substrates at a crime scene. Body fluids collected from crime scenes are not entirely free from substrate microbes whose effects on the microbiome-based identification of body fluids are not well understood. In this study, five body fluids (peripheral blood, menstrual blood, nasal secretions, saliva, and semen) were deposited on sterile swabs, bedspreads, and floors under indoor exposure conditions for 7 days. The microbial communities in the samples were characterized using amplicon sequencing targeted V4 region of 16S rRNA gene. The results showed that the microbial communities of fresh samples deposited on sterile swabs clustered together according to the type of body fluid. The microbial composition of the body fluids deposited on the bedspread and floor is significant different from those deposited on sterile swabs. The microbial communities of mock body fluids were a mixture of microbes from pure body fluids and environmental microbes. FEAST analysis showed that the microbes of mock saliva samples were mainly from pure body fluids (51.53 % and 63.04 % on the bedspread and floor, respectively), but not from substrates (25.70 % and 18.92 % on the bedspread and floor, respectively). Contrary results were observed in peripheral blood, mock nasal secretion, and semen samples. All samples were mainly clustered based on the substrate, but not on the type of body fluid in the PCoA visualization. PERMANOVA results showed that the substrate accounted for more of the variance (R = 0.211, P < 0.001) than the type of body fluid (R = 0.152, P < 0.001). MicroDecon was used to remove contamination by microbes from the substrate of mock body fluid samples. PCoA and PERMANOVA were performed using decontaminated data. The results showed that samples were no longer clustered based on the substrate, and the type of body fluid (R = 0.240, P < 0.001) accounted for more of the variance in the microbial communities of samples than the substrate (R = 0.108, P < 0.001). Our results suggest that environmental microbiota from substrates may interfere with the microbiome-based identification of forensically relevant body fluids. To some extent, decontamination could decrease the effects of the substrate on the microbial communities of the samples and enhance the ability to distinguish between the types of body fluids. This pilot study will be valuable in promoting the application of microbiome-based stain analysis in forensics.

We performed an internal laboratory validation of the Precision ID GlobalFiler NGS STR panel v2 kit to assist the introduction of the technology into the routine forensic casework practice. The study was designed and evaluated based not only on the key validation standards like sensitivity, stability, reproducibility, repeatability, mixture, and concordance, but we also tested the effect of reduced input DNA, we measured and applied locus-specific analytical threshold values, tested two different PCR cycle conditions, sequence artifacts and stutters were also analysed. During the study we also tested the new method on real casework samples. The sensitivity study confirmed that adding 500 pg template DNA for library preparation can be optimal at base PCR cycle number (that was 24), because the measured average heterozygote balance was not lower than 0.82, and each allele was detected above the analytical threshold. However, contrary to previous communications, increasing the PCR cycle numbers up to 28 has not resulted the significant elevation of the heterozygote imbalance. According to our results, raised PCR cycle condition (i.e. 28) is appropriate at or below 150 pg total input DNA. For most loci, the calculated AT was lower than the manufacturer's recommended. Applying the newly established ATs with raised PCR cycle conditions the allele detection sensitivity and reliability increased. We observed allele dropouts only at the 15 pg template DNA experiments with 5 % frequency, that is better to previously published studies. This result indicates that this low amount of DNA (i.e. 15 pg) could be a minimum limit of template input for a potentially successful analysis. In the mixture study the minor contributor could be detected up to 1:19 mixture ratio. We detected minor alleles in all measurements and concentrations above the threshold if the template DNA were fixed and only SNP differences were observed between the same alleles of the contributors. To test concordance between the new method and traditional STR genotyping we analysed 58 Hungarian individual samples in parallel. Nearby the detected 248 different length-based alleles on the 31 loci in the sample pool we revealed additional 75 sequence variant alleles, that represent an approximately 23 % increase in the total number of observed alleles. The casework study confirmed that the Precision ID GlobalFiler NGS STR panel v2 kit is effective even in genotyping degraded samples with extremely low levels of DNA, if we apply elevated cycle number for library preparation and use locus-specific analytical thresholds.

Massively Parallel Sequencing (MPS) has gained interest in the forensic community over the past decade. Most of the published MPS methods focus on specialty applications intended for use in a limited number of samples with protocols that are relatively laborious. Recent developments using Reverse-Complement PCR enable an efficient MPS protocol suited for routine analysis of high numbers of samples. This method is implemented in the IDseek® OmniSTR™ Global Autosomal STR Profiling kit (Nimagen) for sequencing 28 of the most commonly used forensic autosomal STRs, one Y-chromosomal STR and Amelogenin. This study describes the validation of this kit and focuses on sensitivity, inhibitor tolerance, sequence variation detection and performance with mixtures up to 5 contributors. Results are compared to a Capillary Electrophoresis method (the PowerPlex® Fusion 6 C system, Promega) and the first commercial forensic MPS kit (ForenSeq™ DNA Signature prep, Qiagen) and for a concordance study with data from the Powerseq® MPS kit as well. Analysis settings in FDSTools are deduced and discussed, and an almost completely automated analysis is achieved. Using FDSTools noise correction, contributions in a mixture down to a level of 1.5 % of the major allele of a marker can be detected.

Biogeographical ancestry analysis is valuable in forensic investigations, especially in missing person cases or crimes without eyewitnesses, as it helps to infer geographic origins from genetic markers. This approach enhances forensic efforts by providing essential clues for identifying individuals with limited direct evidence. Slavic-speaking populations are poorly distinguishable based on human genome variability. However, recent studies show that even populations with close biogeographic origin could be differentiated based on salivary microbiomes. Nevertheless, the salivary microbiomes of Slavs have not been characterized yet. Therefore, this study aimed to compare the composition of the salivary microbiomes of Western and Southern Slavs' representatives. 16S rRNA libraries from salivary microbiomes of 40 Poles (Western Slavs) and 40 Serbians (Southern Slavs) were prepared via PCR and sequenced on the MiSeq FGx platform (Illumina), giving approximately 100,000 reads per sample. Bioinformatic and statistical analyses were performed to assess the alpha and beta diversity of microbiomes and determine the differences in the abundance of bacterial genera between the groups studied. Analyses of alpha (ACE, Chao1, Shannon, and Simpson) and beta (Jaccard and Bray-Curtis dissimilarity) diversities in the salivary microbiomes clearly distinguished between Poles and Serbians. Alpha and beta diversity metrics were significantly higher in the Serbian population. Fusobacterium, Lautropia, Porphyromonas, Actinobacillus, Capnocytophaga, and Kingella were the most significantly increased genera in Serbians, whereas Veillonella, Selenomonas, Megasphaera, and Atopobium were more prevalent in Poles. In summary, our study identified significant differences in the salivary microbiomes of Poles and Serbians, with distinct microbial signatures associated with each population. These findings highlight the potential of salivary microbiome analysis as a tool for predicting biogeographic ancestry. Nevertheless, further analysis extended to other Slavic-speaking populations is necessary to clarify this issue.

Contact or touch DNA traces from stones account for around 5 % of all crime scene-related swab samples analysed in our department. These traces are often used to identify perpetrators in cases such as burglary, when a stone is used as a tool to break a window or in cases of property damage during riots. Provided that a DNA profile can be obtained in such a case, questions may arise in court regarding the possibilities of DNA transfer onto the stone. Was the subject's DNA indeed transferred onto the stone while it was being used for the crime, or was it already present as background DNA? Alternatively, could it have been transferred by other means, such as by handing over the stone to someone else who then threw it, or by touching it during an attempt to prevent someone else from throwing it? This study focused on two scenarios: experiments involving different participants throwing various stones and a handover scenario where one person touched the stone and another person threw it. We observed that the amount of DNA transferred/detected on the stone is mainly dependent on the individual handling it rather than on the properties of the stone itself or on the order in which the stones are thrown. In the handover scenario, the person who first touched the stone was found to be the main contributor to the trace as often as the person who eventually threw the stone. Our findings therefore confirm that no conclusions can be drawn about the way of interaction with the stones based solely on the obtained DNA profiles.

Forensic services worldwide often encounter considerable challenges relating to funding and infrastructure. Smaller jurisdictions or areas where forensic resources are scarce are faced with complicated choices in how they approach criminal casework, with a number of options available. Often these involve trade-offs between cost, time and data quality. Faced with such decisions it becomes important for the field to acknowledge the realities facing such jurisdictions, discuss the pros and cons of each approach, and identify a framework for making such decisions. This novel paper, reviews the available literature and identifies three main solutions for consideration: 1) the use of satellite laboratories for sample triage, 2) the use of a main regional laboratory for full forensic analysis and 3) the use of rapid DNA by police for reducing backlogs. Alongside these strategies, the impacts of cost and quality in regard to each of the stated options are considered. While the literature supports the assertion that some methods can reduce downstream costs via the reduction in turnaround times, there is limited data highlighting the business case used to support decision making when considering these options including the use of cost:benefit analyses or case studies, emphasizing the novelty of this paper. This is likely due to the commercialized nature of the forensic sector preventing the publication of a private laboratory's business approach. The lack of emphasis on the 'business case' in forensic literature has the potential to mislead R&D scientists who may consequently fail to consider such factors when performing their own research.

MicroRNAs (miRNAs) are promising biomarkers for forensic body fluid identification owing to their small size, stability against degradation, and differential expression patterns. However, the expression of most body fluid-miRNAs is relative (differentially expressed in certain body fluids) rather than absolute (exclusively expressed in a specific body fluid). Moreover, different body fluids contain heterogeneous cell types, complicating their identification. Therefore, appropriate normalization strategies to eliminate non-biological variations and robust models to interpret expression levels accurately are necessary prerequisites for applying miRNAs in body fluid identification. In this study, the expression stability of six candidate reference genes (RGs) across five body fluids was validated using geNorm, NormFinder, BestKeeper and RankAggreg, and the most suitable combination of RGs (hsa-miR-484 and hsa-miR-191-5p) was identified under our experimental conditions. Subsequently, we systematically evaluated the expression patterns of the 28 most promising body fluid-specific miRNA markers using TaqMan RT-qPCR and selected the optimal combination of markers (12 miRNAs) to establish a multi-class support vector machine (MSVM) classification model. An independent test set (60 samples) was used to validate the accuracy of the proposed classification model, while an additional 30 casework samples were used to assess its robustness. The MSVM model accurately predicted the body fluid origin for almost all (59/60) single-source samples. Moreover, this model demonstrated the capability to identify aged forensic samples and to predict the primary components of mixed stains to a certain extent. In summary, this study presented a miRNA-based MSVM classification model for forensic body fluid identification using the qPCR platform. However, extensive validation, especially inter-laboratory collaborative exercises, is necessary before miRNA can be routinely applied in forensic identification practice.

Advancements in sequencing and laboratory technologies have enabled forensic genetic analysis on increasingly low quality and degraded DNA samples. However, existing computational methods applied to genotyping and imputation for generating DNA profiles from degraded DNA have not been tested for forensic applications. Here we simulated sequencing data of varying qualities-coverage, fragment lengths, and deamination patterns-from forty individuals of diverse genetic ancestries. We used this dataset to test the performance of commonly used genotype and imputation methods (SAMtools, GATK, ATLAS, Beagle, and GLIMPSE) on five different SNP panels (MPS-plex, FORCE, two extended kinship panels, and the Human Origins array) that are used for forensic and population genetics applications. For genome mapping and variant calling with degraded DNA, we find use of parameters and methods (such as ATLAS) developed for ancient DNA analysis provides a marked improvement over conventional standards used for next generation sequencing analysis. We find that ATLAS outperforms GATK and SAMtools, achieving over 90 % genotyping accuracy for the four largest SNP panels with coverages greater than 10X. For lower coverages, decreased concordance rates are correlated with increased rates of heterozygosity. Genotype refinement and imputation improve the accuracy at lower coverages by leveraging population reference data. For all five SNP panels, we find that using a population reference panel representative of worldwide populations (e.g., the 1000 Genomes Project) results in increased genotype accuracies across genetic ancestries, compared to ancestry-matched population reference panels. Importantly, we find that the low SNP density of commonly used forensics SNP panels can impact the reliability and performance of genotype refinement and imputation. This highlights a critical trade-off between enhancing privacy by using panels with fewer SNPs and maintaining the effectiveness of genomic tools. We provide benchmarks and recommendations for analyzing degraded DNA from diverse populations with widely used genomic methods in forensic casework.

Hair is an important type of biological evidence at crime scenes. However, the highly degraded nature of DNA fragments in hair shafts poses challenges for the detection of nuclear DNA (nuDNA) through capillary electrophoresis-based short tandem repeat (STR) genotyping. In this study, an all-in-one multiplex system named MGIEasy Signature Identification Library Prep Kit (MGI Tech, China) was employed to the simultaneous genotyping of both mitochondrial DNA (mtDNA) and nuDNA in hair shafts. This system is based on massively parallel sequencing (MPS) technology and encompasses Amelogenin, STRs, single nucleotide polymorphisms (SNPs) and mtDNA hypervariable regions (HVRs) in a single reaction. A total of 370 hair shafts, together with 180 blood samples as the references, were examined. The mtDNA analysis of 110 unrelated blood samples unveiled a total of 150 homoplasmic variants and 105 distinct haplotypes, revealing population polymorphisms in the Guangdong Han Chinese. The study also delved into the detection of mtDNA heteroplasmy, revealing 8.18 % and 16.36 % of individuals with point heteroplasmies (PHPs) in blood and hair shaft samples, respectively. Additionally, hair shafts with DNA extracted using the Investigator method yielded higher average depth of coverage (DoC), lower drop-out rate for SNP genotyping, higher nuDNA genotyped rates and success rates, than those using the MinElute method. In the longitudinal research, a gradual decrease in the total DoC of mtDNA fragments was observed along the length of the hair shaft, from the proximal root to the distal end. In contrast, the DoC of nuDNA exhibited a relatively stable pattern along the length of the hair shafts. The study contributes valuable insights into the simultaneous detection of nuDNA and mtDNA in hair shafts, emphasizing the need for optimized DNA extraction and detection methods for these highly degraded samples.

We examine 31,011 PPY23 profiles at the population, metapopulation and world levels. Most haplotypes appear only once but a few have higher counts, including a set of 23 matching profiles in Delhi, India and a set of 16 matching profiles in Burkina Faso with one additional matching American African profile. We estimate Fvalues to be used as "theta" (θ) in match probability calculations, following the method we used in our earlier survey of autosomal STR data. Match probability estimates using Fˆ or the κ method of Brenner for a previously unseen profile are similar but differ for any profile previously seen.

DNA typing is essential for identifying crime scene evidence and missing and unknown persons. Molecular tags historically have been incorporated into DNA typing reactions to improve result interpretation. Molecular tags like barcodes and unique identifiers are integral to MPS, aiding in sample tracking and error detection. However, these tags do not fully leverage sequence variation to enhance quality control. To address this need, molecular etches, which are synthetic oligonucleotides that serve as an internal molecular information management system, are introduced. Molecular etches encode detailed sample information improving sample workflow history, tracking, contamination detection, and authenticity verification. Validation studies demonstrate the robustness of molecular etches in genomic sequencing, making them a valuable quality tool for forensic DNA analysis.

Research with human genetic data or human beings more generally requires valid informed consent. However, several exceptions exist to this universal principle, usually for situations where it is impossible or at least impractical to obtain this consent. Those exceptions are usually bound to requirements of necessity, shared benefit for the concerned community and minimization of harmful impact. Research without consent is normally subject to approval by an ethics review board. However, secondary use for research may also be based on statutory research privileges in the law. We identified several legal provisions in different countries, permitting the secondary use of DNA data that has been collected without consent for law enforcement purposes, which raises ethical questions. Most of the respective laws seem to be connected to privacy rules and permit the use of de-identified data only. However, whether individual DNA profiles in suspect and offender databases can actually be de-identified must be questioned. What appears to be less critical in terms of re-identification risk are the frequently mentioned "statistical indices", most likely referring to aggregated allele frequencies. However, reflections about data anonymity and a narrow purpose limitation to research aiming at the improvement of calculations of weight of evidence seem not to be the sole reasons for permitting research with entries from criminal offender databases. Distinctions between convicted offenders and other people in the database suggest an assumption of forfeiting some level of privacy protection upon conviction underlying some of these provisions. The use of entries on national DNA databases could therefore amount to problematic additional punishment.

Y-chromosome short tandem repeat (Y-STR) is an important type of genetic markers in the human genome, widely used in molecular anthropology and forensic genetics. However, most Y-STR studies has been focused on the length-based variations resulting from differences in the number of repeat units. Less attention was paid to sequence-based Y-STR variations. Consequently, sequence-based variation characteristics of Y-STRs in Chinese populations remain insufficiently studied. In this study, targeted sequencing of 42 Y-STR loci was performed for 331 Chinese Han males (with an average sequencing depth of 612 ×), unveiling a total of 387 sequence allele types and their frequencies in the population. Repeat pattern variations were observed in seven loci containing multiple repeat units. Across all sequenced repeat and flanking regions, 46 single-nucleotide substitutions and insertion/deletion variations were identified, including 13 mutations not recorded in the dbSNP database. Twenty-seven previously unreported sequence-based alleles were identified. Additionally, differences in Y-STRs between the Chinese Han population and three American populations (African Americans, Caucasians, and Hispanics) were revealed from sequence-based data analysis. In summary, this study provides a detailed summary of the sequence features of 42 Y-STRs in the Chinese Han population, improving our understanding of Y-STRs and providing basic data of sequence variations for the application of Y-STRs.

Powders containing illicit substances are frequently poured into capsules and then distributed in small bags to users, often via intermediaries. We report on the transfer of DNA between individuals involved in making, packing, and transporting capsules within ziplock bags (ZLB) via two pathways, each using 10 ZLBs. A two-person chain was created where participant A made and packed the capsules into ZLBs and participant C then carried the bags for four days. A three-person chain was devised where participant A made the capsules, participant B placed the capsules in bags, and participant C carried the bags. The ZLBs were sampled for DNA on the inside, the inner semi-protected portion of the opening, and the outside surface. The exterior of capsules were also sampled along with a storage container. DNA profiling using Verifiler™ Plus was performed with data deconvoluted by STRmix™. Informative DNA profiles were obtained from capsules despite evidence of DNA transfer from the capsules to both storage containers and the inside of the ZLB. The outside of ZLBs yielded complex mixtures, however, the inside of the bag and the exterior of capsules, which had greater protection, yielded profiles with predominately only one to two contributors. This highlights that the inside of the bags and exterior of capsules could be targeted to identify individuals involved in the early packaging stages of the illicit drug pathway while the outside provided more information on recent handling.