A DNA typing panel of 201 genetic markers for degraded samples: development and validation.

With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.

Evaluation of Storage Conditions and the Effect on DNA from Forensic Evidence Objects Retrieved from Lake Water.

DNA analysis of traces from commonly found objects like knives, smartphones, tapes and garbage bags related to crime in aquatic environments is challenging for forensic DNA laboratories. The amount of recovered DNA may be affected by the water environment, time in the water, method for recovery, transport and storage routines of the objects before the objects arrive in the laboratory. The present study evaluated the effect of four storage conditions on the DNA retrieved from bloodstains, touch DNA, fingerprints and hairs, initially deposited on knives, smartphones, packing tapes, duct tapes and garbage bags, and submerged in lake water for three time periods. After retrieval, the objects were stored either through air-drying at room temperature, freezing at -30 °C, in nitrogen gas or in lake water. The results showed that the submersion time strongly influenced the amount and degradation of DNA, especially after the longest submersion time (21 days). A significant variation was observed in success for STR profiling, while mtDNA profiling was less affected by the submersion time interval and storage conditions. This study illustrates that retrieval from water as soon as possible and immediate storage through air-drying or freezing before DNA analysis is beneficial for the outcome of DNA profiling in crime scene investigations.

A preliminary study on identification of the blood donor in a body fluid mixture using a novel compound genetic marker blood-specific methylation-microhaplotype.

Blood-containing mixtures are frequently encountered at crime scenes involving violence and murder. However, the presence of blood, and the association of blood with a specific donor within these mixtures present significant challenges in forensic analysis. In light of these challenges, this study sought to address these issues by leveraging blood-specific methylation sites and closely linked microhaplotype sites, proposing a novel composite genetic marker known as "blood-specific methylation-microhaplotype". This marker was designed to the detection of blood and the determination of blood donor within blood-containing mixtures. According to the selection criteria mentioned in the Materials and Methods section, we selected 10 blood-specific methylation-microhaplotype loci for inclusion in this study. Among these loci, eight exhibited blood-specific hypomethylation, while the remaining two displayed blood-specific hypermethylation. Based on data obtained from 124 individual samples in our study, the combined discrimination power (CPD) of these 10 successfully sequenced loci was 0.999999298. The sample allele methylation rate (Ram) was obtained from massive parallel sequencing (MPS), which was defined as the proportion of methylated reads to the total clustered reads that were genotyped to a specific allele. To develop an allele type classification model capable of identifying the presence of blood and the blood donor, we used the Random Forest algorithm. This model was trained and evaluated using the Ram distribution of individual samples and the Ram distribution of simulated shared alleles. Subsequently, we applied the developed allele type classification model to predict alleles within actual mixtures, trying to exclude non-blood-specific alleles, ultimately allowing us to identify the presence of blood and the blood donor in the blood-containing mixtures. Our findings demonstrate that these blood-specific methylation-microhaplotype loci have the capability to not only detect the presence of blood but also accurately associate blood with the true donor in blood-containing mixtures with the mixing ratios of 1:29, 1:19, 1:9, 1:4, 1:2, 2:1, 7:1, 8:1, 31:1 and 36:1 (blood:non-blood) by DNA mixture interpretation methods. In addition, the presence of blood and the true blood donor could be identified in a mixture containing four body fluids (blood:vaginal fluid:semen:saliva = 1:1:1:1). It is important to note that while these loci exhibit great potential, the impact of allele dropouts and alleles misidentification must be considered when interpreting the results. This is a preliminary study utilising blood-specific methylation-microhaplotype as a complementary tool to other well-established genetic markers (STR, SNP, microhaplotype, etc.) for the analysis in blood-containing mixtures.

Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics.

OBJECTIVES: To establish and forensically verify a 42 microhaplotypes (mircohaps, MHs) multiplex assay system based on next-generation sequencing (NGS), and to explore the application value of this system in the practice of forensic genetics. METHODS: A total of 42 highly polymorphic MHs were selected from previous studies, and sequenced by the MiSeq FGxTM platform to verify the repeata-bility, sensitivity, specificity, stability, and mixture analysis ability of the detection system. Through population genetic investigation of 102 unrelated Chinese Han individuals in Liyang City, Jiangsu Province, China, the application value of this system in forensic genetics was evaluated. RESULTS: The sequencing repeatability of the 42-plex MHs assay was 100% and the sensitivity was as low as 0.062 5 ng. The system had the ability to withstand the interference of indigo (≤2 500 ng/µL), humic acid (≤9 ng/µL), hemoglobin(≤20 µmol), and urea (≤200 ng/µL) and to detect mixtures of 2 people (1∶19), 3 people (1∶1∶9) and 4 people (1∶1∶1∶9). Based on 102 individual data, the combined power of discrimination and the combined power of exclusion were 1-3.45×10-30 and 1-3.77×10-11, respectively, and the average effect value of alleles was 2.899. CONCLUSIONS: The 42-plex MHs assay was successfully established in this study and this system has high repeatability and sensitivity, good anti-jamming ability and mixture analysis ability. The 42 MHs are highly polymorphism and have good application value in individual identification and paternity testing.

Analysis of 26 STR loci (PowerPlex® Fusion 6C System) in a mestizo population from Mexico city.

BACKGROUND: Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes to enable laboratories to use these genetic systems to interpret and solve forensic casework. However, in Mexico, there are no studies with the PowerPlex Fusion 6C System, which includes 26 STRs (23 autosomal STRs and 3 Y-STRs). METHODS AND RESULTS: 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 312 different alleles were observed. Combined PE and PD were 99.999999809866% and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. CONCLUSIONS: The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied and justify its possible application in other Mexican Mestizo populations.

Comparison of DNA recovery methods and locations from regularly-worn hooded jumpers before and after use by a second wearer.

Items of worn clothing are routinely examined for DNA in forensic casework, commonly with the expectation that at least some of the DNA will come from a wearer of the item, so-called 'wearer DNA'. This study investigated DNA recovered from hooded jumpers that were regularly worn and laundered for four weeks and then subsequently worn by a different individual for four hours. This study also systematically investigated whether using different recovery methods or sampling locations on the jumpers might distinguish between DNA deposited by the regular and most recent wearers of clothing. Four volunteers each wore a new hooded jumper regularly (6 h/day, 2 days/week, washed at weekends) during two 4-week periods. At the end of each month, DNA was first recovered by cutting out and mini-taping the inside left cuff, half-collar, pocket and underarm fabric. The jumpers were then worn by a different individual for four hours, and DNA was again recovered by cutting out and mini-taping, but this time from the inside right cuff, half-collar, pocket and underarm fabric. All DNA samples (n = 128) were quantified and profiled. DNA quantities ranged from 0 to âˆ¼40 ng with an outlier of âˆ¼150 ng, and no significant differences were observed among recovery methods and sampling locations, nor whether one or two wearers had worn the jumpers. However, one volunteer consistently deposited significantly more DNA to their jumpers than two other volunteers, confirming the impact of 'shedder status' on DNA deposition during wearing of clothing. When jumpers were regularly worn by one wearer, the majority (72.7-83.3 %) of the samples for all wearers across both months comprised a major profile of the wearer with a minor profile of non-wearer alleles. When jumpers were then worn by a second wearer, the composition of the profiles obtained were generally reproducible across the recovery methods used, the sampling locations and the two replicates of the experiment for each pairing of wearers. However, profile compositions differed between wearer pairings. Overall, ∼60 % of profiles obtained gave a major profile of the regular wearer, whereas âˆ¼30 % gave a major profile of the second wearer. The remaining profiles comprised other much less frequent observations of single-source profiles of each wearer and equal proportions of DNA from both wearers. Non-wearer DNA was also observed in the majority of samples, both before and after jumpers were worn by a second wearer. For one volunteer's jumpers, a recurring non-wearer DNA profile was observed that could be attributed to their romantic partner, and this DNA persisted on the jumpers even after being worn by the second wearer. This study provides insight on the impact of shedder status, multiple wearers, different recovery methods and sampling locations on the quantities of DNA and compositions of DNA profiles recovered from authentically regularly-worn hooded jumpers. The findings also provide a preliminary dataset that can be used to infer activity level probabilities in casework.

Genomic structure analysis and construction of DNA fingerprint for four sheep populations.

The Yongdeng Qishan sheep (QS) is a sheep population found locally in China. To gain in-depth knowledge of its population characteristics, three control groups were chosen, comprising the Lanzhou fat-tailed sheep (LFT), TAN sheep (TAN), and Minxian black fur sheep (MBF), inhabiting the nearby environments. This study genotyped a total of 120 individuals from four sheep populations: QS, LFT, TAN, and MBF. Using Specific-Locus Amplified Fragment Sequencing, we conducted genetic diversity, population structure, and selective sweep analysis, and constructed the fingerprint of each population. In total, there were 782 535 single nucleotide polymorphism (SNP) variations identified, with most being situated within regions that are intergenic or intronic. The genetic diversity analysis revealed that the QS population exhibited lower genetic diversity compared to the other three populations. Consistent results were obtained from the principal component, phylogenetic tree, and population structure analysis, indicating significant genetic differences between QS and the other three populations. However, a certain degree of differentiation was observed within the QS population. The linkage disequilibrium (LD) patterns among the four populations showed clear distinctions, with the QS group demonstrating the most rapid LD decline. Kinship analysis supported the findings of population structure, dividing the 90 QS individuals into two subgroups consisting of 23 and 67 individuals. Selective sweep analysis identified a range of genes associated with reproduction, immunity, and adaptation to high-altitude hypoxia. These genes hold potential as candidate genes for marker-assisted selection breeding. Additionally, a total of 86 523 runs of homozygosity (ROHs) were detected, showing non-uniform distribution across chromosomes, with chromosome 1 having the highest coverage percentage and chromosome 26 the lowest. In the high-frequency ROH islands, 79 candidate genes were associated with biological processes such as reproduction and fat digestion and absorption. Furthermore, a DNA fingerprint was constructed for the four populations using 349 highly polymorphic SNPs. In summary, our research delves into the genetic diversity and population structure of QS population. The construction of DNA fingerprint profiles for each population can provide valuable references for the identification of sheep breeds both domestically and internationally.

Assessment of Blood and Semen Detection and DNA Collection from Swabs up to Three Months after Deposition on Five Different Cloth Materials.

Body fluid identification plays a crucial role in criminal investigations. Because of their presence in many cases, blood and semen are the most relevant body fluids in forensic sciences. Based on antigen-antibody reactions binding unique proteins for each body fluid, serological assays represent one of the most rapid and highly specific tests for blood and semen. Currently, few studies have assessed the factors affecting body fluid identification by applying these assays. This work aimed to study the effect of different fabrics from clothes and time since deposition on identification through immunochromatographic tests for blood and semen, DNA isolation, and STR profiling from these samples. Body fluids were deposited on black- and white-dyed denim and cotton fabrics, and on leather. Afterward, blood and semen were sampled at 1 day, 30 days, and 90 days after deposition and identified by using the SERATEC® HemDirect Hemoglobin Test and the PSA Semiquant and SERATEC® BLOOD CS and SEMEN CS tests, respectively. Laboratory and crime scene tests presented similar performances for the detection of blood and semen stains on every tested fabric. No differences were found on band intensities between timepoints for all fabrics. It was possible to recover and identify blood and semen samples up to three months after deposition and to obtain full STR profiles from all the tested fabrics. Both body fluid STR profiles showed differences in their quality between 1 and 90 days after deposition for all fabrics except for black cotton for semen samples. Future research will expand the results, assessing body fluid identification on other substrates and under different environmental conditions.

Technical Note: A simple FTA® based method for the direct STR amplification of human foetal tissues.

Short tandem repeats (STRs) or microsatellites are short, tandemly repeated DNA sequences that involve a repetitive unit of 1-6 bp. DNA isolation and purification from a large number and often compromised samples gives problems to forensic labs for STR typing. Many of the conventional methods used in the isolation and purification of DNA from forensic samples are time consuming, expensive, hazardous for health and are often associated with greater risks of cross contamination. FTA® technology is a method designed to simplify the collection, shipment, archiving and purification of nucleic acid from a wide variety of biological samples. We report a new method for the direct STR amplification which can amplify STR loci from human foetal tissues spotted on FTA cards, bye-passing the need of DNA purification. The STR loci amplified by this method was compared with conventional method of STR profiling and was found absolutely matching. Therefore, this new method is demonstrated to be very useful for fast, less expensive and non- hazardous forensic DNA analysis.

Population genetic analyses of Eastern Chinese Han nationality using ForenSeq™ DNA Signature Prep Kit.

Currently, the most commonly used method for human identification and kinship analysis in forensic genetics is the detection of length polymorphism in short tandem repeats (STRs) using polymerase chain reaction (PCR) and capillary electrophoresis (CE). However, numerous studies have shown that considerable sequence variations exist in the repeat and flanking regions of the STR loci, which cannot be identified by CE detection. Comparatively, massively parallel sequencing (MPS) technology can capture these sequence differences, thereby enhancing the identification capability of certain STRs. In this study, we used the ForenSeq™ DNA Signature Prep Kit to sequence 58 STRs and 94 individual identification SNPs (iiSNPs) in a sample of 220 unrelated individuals from the Eastern Chinese Han population. Our aim is to obtain MPS-based STR and SNP data, providing further evidence for the study of population genetics and forensic applications. The results showed that the MPS method, utilizing sequence information, identified a total of 486 alleles on autosomal STRs (A-STRs), 97 alleles on X-chromosome STRs (X-STRs), and 218 alleles on Y-chromosome STRs (Y-STRs). Compared with length polymorphism, we observed an increase of 260 alleles (157, 31, and 72 alleles on A-STRs, X-STRs, and Y-STRs, respectively) across 36 STRs. The most substantial increments were observed in DYF387S1 and DYS389II, with increases of 287.5% and 250%, respectively. The most increment in the number of alleles was found at DYF387S1 and DYS389II (287.5% and 250%, respectively). The length-based (LB) and sequence-based (SB) combined random match probability (RMP) of 27 A-STRs were 6.05E-31 and 1.53E-34, respectively. Furthermore, other forensic parameters such as total discrimination power (TDP), cumulative probability of exclusion of trios (CPEtrio), and duos (CPEduo) were significantly improved when using the SB data, and informative data were obtained for the 94 iiSNPs. Collectively, these findings highlight the advantages of MPS technology in forensic genetics, and the Eastern Chinese Han genetic data generated in this study could be used as a valuable reference for future research in this field.

The impact of substrate characteristics on the collection and persistence of biological materials, and their implications for forensic casework.

This study assessed the level of nucleic acid persistence on the substrate pre-, and post-swabbing, in order to assess whether biological materials (touch, saliva, semen, and blood) are collected differently depending on the substrate characteristics. A total of 48 samples per deposit and substrate variety (n = 384) were assessed by tracking the persistence of nucleic acid using Diamond™ Nucleic Acid Dye (DD) staining and Polilight photography. The number of DD nucleic acid fluorescent complexes formed post-staining were counted (fluorescent count) and in conjunction with the fluorescence signal intensity (DD nucleic acid complex accumulation) used to estimate the level of nucleic acid persistence on substrates. Touch deposits have shown to be the most persistent deposit with strong adhesion capabilities on both substrate verities. Saliva displayed a higher persistence than semen and/or blood. Semen displayed a high collection efficiency as well as a high fluorescence signal intensity. Blood displayed a low persistence on both substrates with a superior collection efficiency that may also indicate a higher probability to become dislodged from surfaces given a particular activity. Our research has shown that the persistence and recovery of biological deposits is not only measurable but more importantly, may have the potential to be estimated, as such, may build an understanding that can provide valuable guidance for collection efficiency evaluations, and the assessing of the probability of particular profiles, given alternate propositions of means of transfer occurring.

Trace DNA Transfer in Co-Working Spaces: The Importance of Background DNA Analysis.

The presence of background DNA (bgDNA) can hinder the evaluation of DNA evidence at the activity level, especially when the suspect is expected to be retrieved due to their habitual occupation of the investigated environment. Based on real-life casework circumstances, this study investigates the prevalence, composition, origin, and probable transfer routes of bgDNA found on personal items in situations where their owner and person of interest (POI) share the same workspace. Baseline values of bgDNA were evaluated on the participants' personal items. Secondary and higher degree transfer scenarios of non-self DNA deposition were also investigated. The DNA from co-workers and co-inhabiting partners can be recovered from an individual's personal belongings. Non-self DNA present on the hands and deposited on a sterile surface can generate uninformative profiles. The accumulation of foreign DNA on surfaces over time appears to be crucial for the recovery of comparable profiles, resulting in detectable further transfer onto other surfaces. For a thorough evaluation of touch DNA traces at the activity level, it is necessary to collect information not only about DNA transfer probabilities but also about the presence of the POI as part of the 'baseline' bgDNA of the substrates involved.

Performance of a 74-Microhaplotype Assay in Kinship Analyses.

Microhaplotypes (MHs) consisting of multiple SNPs and indels on short stretches of DNA are new and interesting loci for forensic genetic investigations. In this study, we analysed 74 previously defined MHs in two of the populations that our laboratory provides with forensic genetic services, Danes and Greenlanders. In addition to the 229 SNPs that originally made up the 74 MHs, 66 SNPs and 3 indels were identified in the two populations, and 45 of these variants were included in new definitions of the MHs, whereas 24 SNPs were considered rare and of little value for case work. The average effective number of alleles (Ae) was 3.2, 3.0, and 2.6 in Danes, West Greenlanders, and East Greenlanders, respectively. High levels of linkage disequilibrium were observed in East Greenlanders, which reflects the characteristics of this population that has a small size, and signs of admixture and substructure. Pairwise kinship simulations of full siblings, half-siblings, first cousins, and unrelated individuals were performed using allele frequencies from MHs, STRs and SNPs from Danish and Greenlandic populations. The MH panel outperformed the currently used STR and SNP marker sets and was able to differentiate siblings from unrelated individuals with a 0% false positive rate and a 1.1% false negative rate using an LR threshold of 10,000 in the Danish population. However, the panel was not able to differentiate half-siblings or first cousins from unrelated individuals. The results generated in this study will be used to implement MHs as investigative markers for relationship testing in our laboratory.

Utilizing Massively Parallel Sequencing (MPS) of Human Leukocyte Antigen (HLA) Gene Polymorphism to Assess Relatedness in Deficiency Parentage Testing.

In the realm of DNA testing with legal implications, the reliability and precision of genetic markers play a pivotal role in confirming or negating paternity claims. This study aimed to assess the potential utility of human leukocyte antigen (HLA) gene polymorphism through massively parallel sequencing (MPS) technology as robust forensic markers for parentage testing involving genetic deficiencies. It sought to redefine the significance of HLA genes in this context. Data on autosomal short tandem repeat (aSTR) mutational events across 18 paternity cases involving 16 commonly employed microsatellite loci were presented. In instances where traditional aSTR analysis failed to establish statistical certainty, kinship determination was pursued via HLA genotyping, encompassing the amplification of 17 linked HLA loci. Within the framework of this investigation, phase-resolved genotypes for HLA genes were meticulously generated, resulting in the definition of 34 inherited HLA haplotypes. An impressive total of 274 unique HLA alleles, which were classified at either the field 3 or 4 level, were identified, including the discovery of four novel HLA alleles. Likelihood ratio (LR) values, which indicated the likelihood of the observed data under a true biological relationship versus no relationship, were subsequently calculated. The analysis of the LR values demonstrated that the HLA genes significantly enhanced kinship determination compared with the aSTR analysis. Combining LR values from aSTR markers and HLA loci yielded conclusive outcomes in duo paternity cases, showcasing the potential of HLA genes and MPS technology for deeper insights and diversity in genetic testing. Comprehensive reference databases and high-resolution HLA typing across diverse populations are essential. Reintegrating HLA alleles into forensic identification complements existing markers, creating a potent method for future forensic analysis.

Trace DNA and its persistence on various surfaces: A long term study investigating the influence of surface type and environmental conditions - Part one, metals.

It is imperative for proper evidence triage that forensic biologists understand what kind of results to expect from certain evidence types submitted for DNA analysis. The persistence of trace DNA has been insufficiently investigated and there is little data available pertaining to the persistence of DNA in different environmental conditions and on different materials. The goal of this study is to increase the available data on this topic which would, in turn, help forensic biologists manage expectations when submitting specific evidence types for DNA testing. The work presented herein is a large-scale persistence project aimed to identify trends in the persistence of trace DNA and indicate 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 7 metals over the course of 1 year (27 time points) under 3 different environmental storage conditions. The results of this experiment show that metal type greatly influences DNA persistence. For instance, copper exhibited an expected poor DNA persistence (up to 4 h) which a purification step did not help increase the DNA yield. Alternatively, DNA can persist for up to a year on lead at levels potentially high enough to allow for forensic DNA testing. Additionally, this study showed that the sample storage environment had no impact on DNA persistence in most cases. When considering DNA type, cfDNA was shown to persist for longer than cellular DNA and persistence as a whole appears to be better when DNA is deposited as mixtures over when deposited alone. Unsurprisingly, it can be expected that DNA recovery rates from trace deposits will decrease over time. However, DNA decay is highly dependent on the metal surface and extremely variable at short time points but slightly less variable as time since deposition increases. This data is intended to add to our understanding of DNA persistence and the factors which affect it.

Predicting probative levels of touch DNA on tapelifts using Diamond™ Nucleic Acid Dye.

Tapelifting is a common strategy to recover touch DNA deposits from porous exhibits in forensic DNA casework. However, it is known that only about 30 % of tapelifts submitted for DNA analysis in operational forensic laboratories yield profiles suitable for comparison or upload to a searchable database. A reliable means to identify and remove non-probative tapelifts from the workflow would reduce sample backlogs and provide significant cost savings. We investigated whether the amount of macroscopic or microscopic fluorescence on a tapelift following staining with Diamond Nucleic Acid Dye (DD), determined using a Polilight and Dino Lite microscope respectively, could predict the DNA yield and/or the DNA profiling outcome using controlled (saliva), semi-controlled (finger mark) and uncontrolled (clothing) samples. Both macroscopic and microscopic DD fluorescence could predict DNA yield and profiling outcome for all sample types, however the predictive power deteriorated as the samples became less controlled. For tapelifts of clothing, which are operationally relevant, Polilight fluorescence scores were significantly impacted by clothing fibres and other non-cellular debris and could not be used to identify non-probative samples. The presence of less than 500 cells on a clothing tapelift using microscopic counting of stained corneocytes was identified as a potential threshold for a non-probative DNA profiling outcome. A broader examination of the reliability of this threshold using a casework trial is recommended. Due to the labour intensiveness of microscopic cell counting, and the increased risk of inadvertent contamination, automation of this process using image software in conjunction with artificial neural networks (ANN) should be explored.

Investigative use of human environmental DNA in forensic genetics.

Individuals leave behind traces of their DNA wherever they go. DNA can be transferred to surfaces and items upon touch, can be released into the air, and may be deposited in indoor dust. The mere presence of individuals in a location is sufficient to facilitate either direct or indirect DNA transfer into the surrounding environment. In this study, we analyzed samples recovered from commonly touched surfaces such as light switches and door handles in an office environment. We evaluated two different methods to isolate DNA and co-extract DNA and RNA from the samples. DNA profiles were compared to the references of the inhabitants of the different locations and were analyzed taking into consideration the type of sampled surface, sampling location and information about the activities in a room during the sampling day. Results from DNA samples collected from surfaces were also compared to those from air and dust samples collected in parallel from the same areas. We characterized the amount and composition of DNA found on various surfaces and showed that surface DNA sampling can be used to detect occupants of a location. The results also indicate that combining information from environmental samples collected from different DNA sources can improve our understanding of DNA transfer events in an indoor setting. This study further demonstrates the potential of human environmental DNA as an investigative tool in forensic genetics.

The Missing Person problem through the lens of information theory.

Missing person cases typically require a genetic kinship test to determine the relationship between an unidentified individual and the relatives of the missing person. When not enough genetic evidence has been collected the lack of statistical power of these tests might lead to unreliable results. This is particularly true when just a few distant relatives are available for genotyping. In this contribution, we considered a Bayesian network approach for kinship testing and proposed several information theoretic metrics in order to quantitatively evaluate the information content of pedigrees. We show how these statistics are related to the widely used likelihood ratio values and could be employed to efficiently prioritize family members in order to optimize the statistical power in missing person problems. Our methodology seamlessly integrates with Bayesian modeling approaches, like the GENis platform that we have recently developed for high-throughput missing person identification tasks. Furthermore, our approach can also be easily incorporated into Elston-Stewart forensic frameworks. To facilitate the application of our methodology, we have developed the forensIT package, freely available on CRAN repository, which implements all the methodologies described in our manuscript.

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.

Nails as optimal source of DNA for molecular identification of 5 decomposed bodies recovered from seawater: from Y-ancestry to personal identification.

Molecular identification of extremely compromised human remains in forensic field is usually performed from DNA typing of bones, which are a difficult sample to work with. Moreover, autosomal STR profiles do not always result in the identification of the donor due to lack of comparisons or non-hit throughout database searching. An attempt to overcome these issues is represented by fingernails as an alternative DNA source and Y-STRs typing to infer both geographical and familial ancestry of the unknown donor. In this study, we analyzed both 24 autosomal and 27 Y-chromosome STRs from unidentified human remains (UHRs) of five males recovered from the water near the southwestern coast of Sardinia by the Italian Harbor Master's Office. Nail clippings provided an optimal source of autologous DNA for molecular identification in a very short time, producing complete autosomal and Y-STR profiles even under conditions of high body degradation. Unfortunately, no match neither compatibility occurred using autosomal STRs (aSTRs), initially. Upon analyzing the Y-haplotypes, we found out they had already been observed in northern Africa, providing us important investigative leads. This prompted the International Criminal Police Organization (INTERPOL) to provide us with references of alleged relatives that were then confirmed to be related. The use of fingernails represents an excellent DNA source especially for genetic identification of decomposed bodies recovered in seawater environment. Notably, DNA extracted from nails gave high-quality Y-STR haplotypes by which predicting paternal ancestry of the unidentified donors may result fundamental in the forensic investigative context.