Profiling age and body fluid DNA methylation markers using nanopore adaptive sampling.

DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research.

Machine learning applications in forensic DNA profiling: A critical review.

Machine learning (ML) is a range of powerful computational algorithms capable of generating predictive models via intelligent autonomous analysis of relatively large and often unstructured data. ML has become an integral part of our daily lives with a plethora of applications, including web, business, automotive industry, clinical diagnostics, scientific research, and more recently, forensic science. In the field of forensic DNA, the manual analysis of complex data can be challenging, time-consuming, and error-prone. The integration of novel ML-based methods may aid in streamlining this process while maintaining the high accuracy and reproducibility required for forensic tools. Due to the relative novelty of such applications, the forensic community is largely unaware of ML capabilities and limitations. Furthermore, computer science and ML professionals are often unfamiliar with the forensic science field and its specific requirements. This manuscript offers a brief introduction to the capabilities of machine learning methods and their applications in the context of forensic DNA analysis and offers a critical review of the current literature in this rapidly developing field.

Operationalisation of the ForenSeq® Kintelligence Kit for Australian unidentified and missing persons casework.

Single nucleotide polymorphism (SNP) genotyping technologies can generate investigative leads for human remains identification, including estimation of biological sex, biogeographical ancestry (BGA), externally visible characteristics (EVCs), identity, uniparental lineage and extended kinship. The ForenSeq® Kintelligence Kit provides forensic laboratories with the ability to apply this suite of genetic tools to forensic samples using one panel targeting 10,230 SNPs (including 56 ancestry-informative, 24 phenotype-informative, 94 identity-informative, 106 X chromosome, 85 Y chromosome and 9867 kinship-informative SNPs) sequenced on the MiSeq FGx® Sequencing System. The ForenSeq® Kintelligence Kit has been internally validated, optimised and operationalised by the Australian Federal Police National DNA Program for Unidentified and Missing Persons (AFP Program) for coronial casework. The internal validation was conducted according to the Scientific Working Group on DNA Analysis Methods guidelines (excluding mixture analysis), focussing on sample types typically encountered in human remains identification casework, such as bones, teeth, nail, blood and hair. The workflow was optimised for a high throughput library preparation and sequencing workflow, and additional analytical thresholds were developed to improve genotyping accuracy for low DNA input samples. Additionally, the genetic intelligence generated from the kit was compared to the self-declared biological sex, EVCs and BGA of the DNA donors to assess concordance. The kit was able to produce high quality SNP profiles from 1.0 ng down to 0.1 ng of DNA, with high repeatability and reproducibility, and minimal background noise. The prediction accuracy for biological sex (95%), hair colour (58%), eye colour (74%) and BGA inferences (consistent: 74%; partially consistent: 10%; inconclusive: 16%) was determined based on self-declared data. Additionally, SNP profiles from a volunteer family group of ten related individuals were uploaded to GEDmatch PRO™ to assess kinship accuracy. The kit was capable of detecting (97%) and accurately classifying (90%) genetic relationships spanning from first to fifth degree. The Kintelligence Kit provides the AFP Program with a robust and reliable genetic intelligence tool for unidentified and missing persons investigations, which has been designed to sequence multiple challenging samples in a single multiplexed assay using existing laboratory instrumentation.

Estimating the time of human decomposition based on skeletal muscle biopsy samples utilizing an untargeted LC-MS/MS-based proteomics approach.

Accurate estimation of the postmortem interval (PMI) is crucial in forensic medico-legal investigations to understand case circumstances (e.g. narrowing down list of missing persons or include/exclude suspects). Due to the complex decomposition chemistry, estimation of PMI remains challenging and currently often relies on the subjective visual assessment of gross morphological/taphonomic changes of a body during decomposition or entomological data. The aim of the current study was to investigate the human decomposition process up to 3 months after death and propose novel time-dependent biomarkers (peptide ratios) for the estimation of decomposition time. An untargeted liquid chromatography tandem mass spectrometry-based bottom-up proteomics workflow (ion mobility separated) was utilized to analyse skeletal muscle, collected repeatedly from nine body donors decomposing in an open eucalypt woodland environment in Australia. Additionally, general analytical considerations for large-scale proteomics studies for PMI determination are raised and discussed. Multiple peptide ratios (human origin) were successfully proposed (subgroups < 200 accumulated degree days (ADD), < 655 ADD and < 1535 ADD) as a first step towards generalised, objective biochemical estimation of decomposition time. Furthermore, peptide ratios for donor-specific intrinsic factors (sex and body mass) were found. Search of peptide data against a bacterial database did not yield any results most likely due to the low abundance of bacterial proteins within the collected human biopsy samples. For comprehensive time-dependent modelling, increased donor number would be necessary along with targeted confirmation of proposed peptides. Overall, the presented results provide valuable information that aid in the understanding and estimation of the human decomposition processes.

Empirical Evidence on Enhanced Mutation Rates of 19 RM-YSTRs for Differentiating Paternal Lineages.

Rapidly mutating Y-chromosomal short tandem repeats (RM Y STRs) with mutation rates ≥ 10-2 per locus per generation are valuable for differentiating amongst male paternal relatives where standard Y STRs with mutation rates of ≤10-3 per locus per generation may not. Although the 13 RM Y STRs commonly found in commercial assays provide higher levels of paternal lineage differentiation than conventional Y STRs, there are many male paternal relatives that still cannot be differentiated. This can be improved by increasing the number of Y STRs or choosing those with high mutation rates. We present a RM Y STR multiplex comprising 19 loci with high mutation rates and its developmental validation (repeatability, sensitivity and male specificity). The multiplex was found to be robust, reproducible, specific and sensitive enough to generate DNA profiles from samples with inhibitors. It was also able to detect all contributor alleles of mixtures in ratios up to 9:1. We provide preliminary evidence for the ability of the multiplex to discriminate between male paternal relatives by analyzing large numbers of male relative pairs (536) separated by one to seven meioses. A total of 96 mutations were observed in 162 meioses of father-son pairs, and other closely related male pairs were able to be differentiated after 1, 2, 3, 4, 5, 6 and 7 meiosis in 44%, 69%, 68%, 85%, 0%, 100% and 100% of cases, respectively. The multiplex offers a noticeable enhancement in the ability to differentiate paternally related males compared with the 13 RM Y STR set. We envision the future application of our 19 RM Yplex in criminal cases for the exclusion of male relatives possessing matching standard Y STR profiles and in familial searching with unknown suspects. It represents a step towards the complete individualization of closely related males.

Touch DNA recovery from unfired and fired cartridges: Comparison of swabbing, tape lifting and soaking.

Over the recent few years, several DNA collection techniques and methodologies have been published for the recovery of DNA from fired cartridge cases. In this study, swabbing, the DNA collection technique currently used in our jurisdiction (NSW, Australia), was compared with tape lifting and soaking to assess DNA recovery rates, DNA quality and profile quality. Brass .22LR and 9mmP cartridges were used as they are the most commonly encountered in our jurisdiction. The cartridges (n = 107) were loaded into cleaned firearm magazines by three volunteers of unknown shedder status, to mimic routine casework sample types. Half of the handled cartridges were fired whilst the other half were kept unfired. STR genotypes were produced at both 29 and 30 PCR cycles to evaluate which improved handler allele detection. DNA recovery rates showed that swabbing recovered significantly less DNA than tape lifting and soaking. Whilst there were no significant differences between tape lifting and soaking, tape lifting, on average, yielded more DNA than soaking. The calibre of ammunition had no influence on DNA recovery and in line with expectations, firing was found to decrease DNA recovery for all three sampling techniques. Assessment of DNA quality showed no evidence of PCR inhibition in any of the samples for this study. However, degradation indices showed that most samples were slightly to moderately degraded. Fewer handler alleles were detected from both fired tape lifted and soaked cartridges than unfired cartridges. Whilst 30 amplification cycles allowed for the detection of slightly more handler alleles, no statistically significant differences were found between 29 and 30 PCR cycles. Nonetheless, 50% of the profiles from unfired soaked cartridges that were non-uploadable after 29 cycles were uploadable after 30 cycles. Furthermore, 83% of profiles from unfired cartridges that were tape lifted were uploadable onto our jurisdiction's database at both 29 and 30 PCR cycles. All magazine controls, despite cleaning, contained some level of background DNA. Furthermore, increasing the number of PCR cycles to 30 also increased the detection of non-handler alleles in DNA profiles. Our results suggest tape lifting yields more uploadable profiles from unfired and fired cartridge cases than swabbing but also more adventitious (non-handler) alleles. However additional research will be needed to evaluate the full potential of this method.

Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing.

DNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a consensus approach, METEORE ( https://github.com/comprna/METEORE ), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are also provided for reproducibility and to enable the systematic application of our analyses to other datasets.

Comparison of Genome-Wide Association Scans for Quantitative and Observational Measures of Human Hair Curvature.

Previous genetic studies on hair morphology focused on the overall morphology of the hair using data collected by self-report or researcher observation. Here, we present the first genome-wide association study (GWAS) of a micro-level quantitative measure of hair curvature. We compare these results to GWAS results obtained using a macro-level classification of observable hair curvature performed in the same sample of twins and siblings of European descent. Observational data were collected by trained observers, while quantitative data were acquired using an Optical Fibre Diameter Analyser (OFDA). The GWAS for both the observational and quantitative measures of hair curvature resulted in genome-wide significant signals at chromosome 1q21.3 close to the trichohyalin (TCHH) gene, previously shown to harbor variants associated with straight hair morphology in Europeans. All genetic variants reaching genome-wide significance for both GWAS (quantitative measure lead single-nucleotide polymorphism [SNP] rs12130862, p = 9.5 × 10-09; observational measure lead SNP rs11803731, p = 2.1 × 10-17) were in moderate to very high linkage disequilibrium (LD) with each other (minimum r2 = .45), indicating they represent the same genetic locus. Conditional analyses confirmed the presence of only one signal associated with each measure at this locus. Results from the quantitative measures reconfirmed the accuracy of observational measures.

Population genetic portrait of Pakistani Lahore-Christians based on 32 STR loci.

Phylogenetic relationship and the population structure of 500 individuals from the Christian community of Lahore, Pakistan, were examined based on 15 autosomal short tandem repeats (STRs) using the AmpFℓSTR Identifiler Plus PCR Amplification Kit and our previously published Y-filer kit data (17 Y-STRs) of same samples. A total of 147 alleles were observed in 15 loci and allele 11 at the TPOX locus was the most frequent with frequency value (0.464). The data revealed that the Christian population has unique genetic characteristics with respect to a few unusual alleles and their frequencies relative to the other Pakistani population. Significant deviations from Hardy-Weinberg equilibrium were found at two loci (D13S317, D18S51) after Boneferroni's correction (p ≤ 0.003). The combined power of discrimination, combined power of exclusion and cumulative probability of matching were 0.999999999999999978430815060354, 0.999995039393942 and 2.15692 × 10-17, respectively. On the bases of genetic distances, PCA, phylogenetic and structure analysis Lahore-Christians appeared genetically more associated to south Asian particularly Indian populations like Tamil, Karnataka, Kerala and Andhra Pradesh than rest of global populations.

DNA recovery from unfired and fired cartridge cases: A comparison of swabbing, tape lifting, vacuum filtration, and direct PCR.

The ability to recover trace DNA from fired cartridge cases can help establish important leads regarding the handler of the ammunition. Over recent years, several DNA recovery techniques for fired ammunition have been published. Three techniques of significant interest include tape lifting, direct PCR, and vacuum filtration. This study aimed to compare these to the swabbing method currently employed in our jurisdiction. Brass and nickel cartridges of five different calibres were spiked with 20ng of saliva and subject to DNA collection using all four DNA recovery methods. Unfired and fired cartridges were tested to examine the effects of firing. Swabbing recovered a greater quantity of DNA than vacuum filtration while no significant differences were found between swabbing and tape-lifting. The calibre of ammunition had no effect on DNA recovery. Firing significantly reduced DNA yield from nickel cartridges, while unfired brass cartridges returned less DNA than unfired nickel cartridges. PCR inhibition was not observed in any samples, although degradation indices suggested that most samples were slightly or moderately degraded. Analysis of profiles showed that swabbing and tape lifting resulted in greater numbers of alleles from fired nickel and brass cartridges compared to direct PCR. Samples from nickel cartridges were found to have a greater number of uploadable profiles than samples from brass cartridges. In addition, three mixed profiles were obtained from the single source spiked cartridges as well as evidence of pre-existing DNA on uncleaned cartridges and contaminating alleles on cleaned cartridges. Our results suggest that tape-lifting can be a suitable alternative to swabbing, but that caution must be taken when interpreting profiles from fired cartridge cases as small amounts of DNA not associated with the handling of the cartridges may be present.

Forensic Autosomal Short Tandem Repeats and Their Potential Association With Phenotype.

Forensic DNA profiling utilizes autosomal short tandem repeat (STR) markers to establish identity of missing persons, confirm familial relations, and link persons of interest to crime scenes. It is a widely accepted notion that genetic markers used in forensic applications are not predictive of phenotype. At present, there has been no demonstration of forensic STR variants directly causing or predicting disease. Such a demonstration would have many legal and ethical implications. For example, is there a duty to inform a DNA donor if a medical condition is discovered during routine analysis of their sample? In this review, we evaluate the possibility that forensic STRs could provide information beyond mere identity. An extensive search of the literature returned 107 articles associating a forensic STR with a trait. A total of 57 of these studies met our inclusion criteria: a reported link between a STR-inclusive gene and a phenotype and a statistical analysis reporting a p-value less than 0.05. A total of 50 unique traits were associated with the 24 markers included in the 57 studies. TH01 had the greatest number of associations with 27 traits reportedly linked to 40 different genotypes. Five of the articles associated TH01 with schizophrenia. None of the associations found were independently causative or predictive of disease. Regardless, the likelihood of identifying significant associations is increasing as the function of non-coding STRs in gene expression is steadily revealed. It is recommended that regular reviews take place in order to remain aware of future studies that identify a functional role for any forensic STRs.

Degradation of nuclear and mitochondrial DNA after γ-irradiation and its effect on forensic genotyping.

Forensic genotyping can be impeded by γ-irradiation of biological evidence in the event of radiological crime; that is, criminal activity involving radioactive material. Oxidative effects within the mitochondria of living cells elicits greater damage to mitochondrial DNA (mtDNA) than nuclear DNA (nuDNA) at low doses. This study presents a novel approach for the assessment of nuDNA versus mtDNA damage from a comparison of genotype and quantity data, while exploring likely mechanisms for differential damage after high doses of γ-irradiation. Liquid (hydrated) and dried (dehydrated) whole blood samples were exposed to high doses of γ-radiation (1-50 kilogray, kGy). The GlobalFiler PCR Amplification Kit was used to evaluate short tandem repeat (STR) genotyping efficacy and nuDNA degradation; a comparison was made to mtDNA degradation measured using real-time PCR assays. Each assay was normalized before comparison by calculation of integrity indices relative to unirradiated controls. Full STR profiles were attainable up to the highest dose, although DNA degradation was noticeable after 10 and 25 kGy for hydrated and dehydrated blood, respectively. This was manifested by heterozygote imbalance more than allele dropout. Degradation was greater for mtDNA than nuDNA, as well as for hydrated than dehydrated cells, after equivalent doses. Oxidative effects due to water radiolysis and mitochondrial function are dominant mechanisms of differential damage to nuDNA versus mtDNA after high-dose γ-irradiation. While differential DNA damage was reduced by cell desiccation, its persistence after drying indicates innate differences between nuDNA and mtDNA radioresistance and/or continued oxidative effects within the mitochondria.

Performance of ancestry-informative SNP and microhaplotype markers.

The use of microhaplotypes (MHs) for ancestry inference has added to an increasing number of ancestry-informative markers (AIMs) for forensic application that includes autosomal single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). This study compares bi-allelic and tri-allelic SNPs as well as MH markers for their ability to differentiate African, European, South Asian, East Asian, and American population groups from the 1000 Genomes Phase 3 database. A range of well-established metrics were applied to rank each marker according to the population differentiation potential they measured. These comprised: absolute allele frequency differences (δ); Rosenberg's informativeness for (ancestry) assignment (In); the fixation index (FST); and the effective number of alleles (Ae). A panel consisting of all three marker types resulted in the lowest mean divergence per population per individual (MDPI = 2.16%) when selected by In. However, when marker types were not mixed, MHs were the highest performing markers by most metrics (MDPI < 4%) for differentiation between the five continental populations.

Ancestry informative markers (AIMs) for Korean and other East Asian and South East Asian populations.

Inference of ancestry from biological evidence can provide investigative information, especially for unknown DNA donors. Although tools for predicting ancestry have been developing, ancestry research focusing on populations relevant for South Korea is not common and markers are seldom chosen specifically to differentiate Koreans from other East Asian and South East Asian populations. Here, we report ancestry informative markers (AIMs) for distinguishing six East/South East Asian regional populations: China, Japan, Indonesia, Philippines, South Korea and Thailand. Individual genotypes from these six populations were available in PanSNPdb: The HUGO Pan-Asian SNP Database. To select AIMs, we calculated four population divergence metrics for each SNP: Nei's FST, Rosenberg's Informativeness (In), the average absolute allele frequency difference between populations (δFmean) and the maximum allele frequency difference between populations (δFmax). Based on these values, we selected 100 single nucleotide polymorphisms (SNPs) for distinguishing the six populations, 13 of which exhibited large allele frequency differences between Koreans and non-Koreans. To assess the performance of the AIMs, we performed principal coordinates analysis (PCoA) on the individuals from all six populations and inferred ancestral population clusters using the STRUCTURE program. In conclusion, we found that the selected AIMs can be applied to distinguish the six East/South East Asian groups and we suggest the markers in this study will be helpful to establish ancestry panels for Korea and neighbouring populations.

Genetic analysis of 12 X-STRs for forensic purposes in Liaoning Manchu population from China.

X-chromosomal short tandem repeats (X-STRs) have been widely used in forensic practice involving complicated cases of kinship and also play an increasingly important role in population genetics. X-STRs have been studied in regional populations of China but there is a lack of data for the Manchu population. In this study, we have investigated the forensic genetic properties of 12 X-STRs in the Investigator Argus X-12 Kit (QIAGEN, Hilden, Germany) in 772 Manchu (male = 514, female = 258) individuals from the Xiuyan and Huanren Manchu autonomous counties of Liaoning province. We observed a total of 166 alleles at 12 X-STR loci with allele frequencies ranging from 0.001295to 0.615285. The most polymorphic locus was DXS10135 with 24 alleles while DXS7423 was the least polymorphic locus with 5 alleles. We found significant linkage disequilibrium (LD) between the following pairs of markers for males: DXS10103/DXS10101, DXS10135/DXS10146, DXS10101/DXS10148, DXS10135/DXS10148, DXS7423/DXS10148 and DXS10079/DXS10148. For females, LD was only observed for DXS10103/DXS10101. The combined power of discrimination was 0.9999999979699 for males and 0.999999999999998 for females. The numbers of observed haplotypes in Manchu males were 310, 172, 182 and 172 in four linkage groups; LG1, LG2, LG3 and LG4, respectively, however, these linkage groups did not form stable haplotypes as indicated by linkage equilibrium (LE) of STRs within the groups and significant LD between the groups. This study represents an extensive report on X-STR marker variation in the Manchu population for forensic applications and population genetic studies.

Bayesian interpretation of discrete class characteristics.

Bayesian interpretation of forensic evidence has become dominated by the likelihood ratio (LR) with a large LR generally considered favourable to the prosecution hypothesis, HP, over the defence hypothesis, HD. However, the LR simply quantifies by how much the prior odds ratio of the probability of HP relative to HD has been improved by the forensic evidence to provide a posterior ratio. Because the prior ratio is mostly neglected, the posterior ratio is largely unknown, regardless of the LR used to improve it. In fact, we show that the posterior ratio will only favour HP when LR is at least as large as the number of things that could possibly be the source of that evidence, all being equally able to contribute. This restriction severely limits the value of evidence to the prosecution when only a single, discrete class characteristic is used to match a subset of these things to the evidence. The limitation can be overcome by examining more than one individual characteristic, as long as they are independent of each other, as they are for the genotypes at multiple loci combined for DNA evidence. We present a criterion for determining how many such characteristics are required. Finally, we conclude that a frequentist interpretation is inappropriate as a measure of the strength of forensic evidence precisely because it only estimates the denominator of the LR.

Dog breed affiliation with a forensically validated canine STR set.

We tested a panel of 13 highly polymorphic canine short tandem repeat (STR) markers for dog breed assignment using 392 dog samples from the 23 most popular breeds in Austria, Germany, and Switzerland. This STR panel had originally been selected for canine identification. The dog breeds sampled in this study featured a population frequency ≥1% and accounted for nearly 57% of the entire pedigree dog population in these three countries. Breed selection was based on a survey comprising records for nearly 1.9 million purebred dogs belonging to more than 500 different breeds. To derive breed membership from STR genotypes, a range of algorithms were used. These methods included discriminant analysis of principal components (DAPC), STRUCTURE, GeneClass2, and the adegenet package for R. STRUCTURE analyses suggested 21 distinct genetic clusters. Differentiation between most breeds was clearly discernable. Fourteen of 23 breeds (61%) exhibited maximum mean cluster membership proportions of more than 0.70 with a highest value of 0.90 found for Cavalier King Charles Spaniels. Dogs of only 6 breeds (26%) failed to consistently show only one major cluster. The DAPC method yielded the best assignment results in all 23 declared breeds with 97.5% assignment success. The frequency-based assignment test also provided a high success rate of 87%. These results indicate the potential viability of dog breed prediction using a well-established and sensitive set of 13 canine STR markers intended for forensic routine use.