Abstract Teeth and bones are frequently the only sources of DNA available for identification of degraded or fragmented human remains. The unique composition of teeth and their location in the jawbone ...provide additional protection to DNA compared to bones making them a preferred source of DNA in many cases. Despite this, post-mortem changes in the structure and composition of teeth, and the location and diagenesis of DNA within them are poorly understood. This review summarises current knowledge of tooth morphology with respect to DNA content and preservation, and discusses the way in which post-mortem changes will affect the recovery of DNA from teeth under a range of commonly used extraction protocols. We highlight the benefits and pitfalls of using specific tooth tissues for DNA extraction and make recommendations for tooth selection and sampling that will maximise DNA typing success. A comprehensive understanding of tooth structure and an appreciation of the relationship between DNA and mineralized tissues in post-mortem teeth are critical for optimal sample selection. More informed sampling methods that target specific tooth tissues will increase the likelihood of successful genetic analysis and allow for efficient and timely missing persons case work and disaster victim identification response.
Massively parallel sequencing following hybridisation enrichment provides new opportunities to obtain genetic data for various types of forensic testing and has proven successful on modern as well as ...degraded and ancient DNA. A customisable forensic intelligence panel that targeted 124 SNP markers (67 ancestry informative markers, 23 phenotype markers from the HIrisplex panel, and 35 Y-chromosome SNPs) was used to examine biogeographic ancestry, phenotype and sex and Y-lineage in samples from different ethnic populations of Pakistan including Pothwari, Gilgit, Baloach, Pathan, Kashmiri and Siraiki. Targeted sequencing and computational data analysis pipeline allowed filtering of variants across the targeted loci. Study samples showed an admixture between East Asian and European ancestry. Eye colour was predicted accurately based on the highest p-value giving overall prediction accuracy of 92.8%. Predictions were consistent with reported hair colour for all samples, using the combined highest p-value approach and step-wise model incorporating probability thresholds for light or dark shade. Y-SNPs were successfully recovered only from male samples which indicates the ability of this method to identify biological sex and allow inference of Y-haplogroup. Our results demonstrate practicality of using hybridisation enrichment and MPS to aid in human intelligence gathering and will open many insights into forensic research in South Asia.
Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA ...analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Furthermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard extraction methods, without the need for specialised equipment or large-volume demineralisation steps.
Massively parallel sequencing can provide genetic data for hundreds to thousands of loci in a single assay for various types of forensic testing. However, available commercial kits require an initial ...PCR amplification of short-to-medium sized targets which limits their application for highly degraded DNA. Development and optimisation of large PCR multiplexes also prevents creation of custom panels that target different suites of markers for identity, biogeographic ancestry, phenotype, and lineage markers (Y-chromosome and mtDNA). Hybridisation enrichment, an alternative approach for target enrichment prior to sequencing, uses biotinylated probes to bind to target DNA and has proven successful on degraded and ancient DNA. We developed a customisable hybridisation capture method, that uses individually mixed baits to allow tailored and targeted enrichment to specific forensic questions of interest. To allow collection of forensic intelligence data, we assembled and tested a custom panel of hybridisation baits to infer biogeographic ancestry, hair and eye colour, and paternal lineage (and sex) on modern male and female samples with a range of self-declared ancestries and hair/eye colour combinations. The panel correctly estimated biogeographic ancestry in 9/12 samples (75%) but detected European admixture in three individuals from regions with admixed demographic history. Hair and eye colour were predicted correctly in 83% and 92% of samples respectively, where intermediate eye colour and blond hair were problematic to predict. Analysis of Y-chromosome SNPs correctly assigned sex and paternal haplogroups, the latter complementing and supporting biogeographic ancestry predictions. Overall, we demonstrate the utility of this hybridisation enrichment approach to forensic intelligence testing using a combined suite of biogeographic ancestry, phenotype, and Y-chromosome SNPs for comprehensive biological profiling.
•Single nucleotide polymorphisms (SNPs) can provide forensic intelligence.•Existing forensic SNP panels are inflexible and may perform poorly on degraded DNA.•We developed and tested a customisable, SNP hybridisation enrichment panel.•The panel provides valuable biological profiles and forensic intelligence.
•Metal surfaces are difficult substrates for trace DNA recovery and amplification.•Metal cations interact with DNA via complex intercalation, irreversible covalent binding and groove ...association.•Five methods of touch DNA sampling include swabbing, tape lifting, soaking, Bardole M-VAC and direct PCR.•There is at most 26% DNA recovery success rate from cartridges, bullets and casings (CBCs).•The surface characteristics of metal substrates are relevant to nucleic acid persistence and recovery.
Trace evidence such as touch (also known as contact) DNA has probative value as a vital forensic investigative tool that can lead to the identification and apprehension of a criminal. While the volume of touch DNA evidence items submitted to forensic laboratories has significantly increased, recovery and amplification of DNA from these items, especially from metal surfaces, remains challenging. Currently little is understood with regards to the underlying mechanisms of metal-DNA interactions in the context of forensic science and how this may impact on DNA recovery. An increased understanding of these mechanisms would allow optimisation of methods to improve outcomes when sampling these materials. This paper reviews the basis of DNA binding to metal substrates, the merits and limitations of current methods and future perspectives of improving recovery and amplification of touch DNA from metal surfaces of forensic interest.
Falsified medicines are a major threat to global health. Antimalarial drugs have been particularly targeted by criminals. As DNA analysis has revolutionized forensic criminology, we hypothesized that ...these techniques could also be used to investigate the origins of falsified medicines. Medicines may contain diverse adventitious biological contamination, and the sealed nature of blister-packages may capture and preserve genetic signals from the manufacturing processes allowing identification of production source(s). We conducted a blinded pilot study to determine if such environmental DNA (eDNA) could be detected in eleven samples of falsified and genuine artesunate antimalarial tablets, collected in SE Asia, which could be indicative of origin. Massively Parallel Sequencing (MPS) was used to characterize microbial and eukaryote diversity. Two mitochondrial DNA analysis approaches were explored to detect the presence of human DNA. Trace eDNA from these low biomass samples demonstrated sample specific signals using two target markers. Significant differences in bacterial and eukaryote DNA community structures were observed between genuine and falsified tablets and between different packaging types of falsified artesunate. Human DNA, which was indicative of likely east Asian ancestry, was found in falsified tablets. This pilot study of the 'pharmabiome' shows the potential of environmental DNA as a powerful forensic tool to assist with the identification of the environments, and hence location and timing, of the source and manufacture of falsified medicines, establish links between seizures and complement existing tools to build a more complete picture of criminal trade routes. The finding of human DNA in tablets raises important ethical issues that need to be addressed.
The human colonization of Remote Oceania remains one of the great feats of exploration in history, proceeding east from Asia across the vast expanse of the Pacific Ocean. Human commensal and ...domesticated species were widely transported as part of this diaspora, possibly as far as South America. We sequenced mitochondrial control region DNA from 122 modern and 22 ancient chicken specimens from Polynesia and Island Southeast Asia and used these together with Bayesian modeling methods to examine the human dispersal of chickens across this area. We show that specific techniques are essential to remove contaminating modern DNA from experiments, which appear to have impacted previous studies of Pacific chickens. In contrast to previous reports, we find that all ancient specimens and a high proportion of the modern chickens possess a group of unique, closely related haplotypes found only in the Pacific. This group of haplotypes appears to represent the authentic founding mitochondrial DNA chicken lineages transported across the Pacific, and allows the early dispersal of chickens across Micronesia and Polynesia to be modeled. Importantly, chickens carrying this genetic signature persist on several Pacific islands at high frequencies, suggesting that the original Polynesian chicken lineages may still survive. No early South American chicken samples have been detected with the diagnostic Polynesian mtDNA haplotypes, arguing against reports that chickens provide evidence of Polynesian contact with pre-European South America. Two modern specimens from the Philippines carry haplotypes similar to the ancient Pacific samples, providing clues about a potential homeland for the Polynesian chicken.
Forensic DNA analysis continues to be hampered by the complex interactions between metals and DNA. Metal ions may cause direct DNA damage, inhibit DNA extraction and polymerase chain reaction (PCR) ...amplification or both. This study evaluated the impact of metal ions on DNA extraction, quantitation, and short tandem repeat profiling using cell‐free and cellular (saliva) DNA. Of the 11 metals assessed, brass exhibited the strongest PCR inhibitory effects, for both custom and Quantifiler Trio quantitation assays. Metal ion inhibition varied across the two quantitative PCR assays and the amount of DNA template used. The Quantifiler Trio internal PCR control (IPC) only revealed evidence of PCR inhibition at higher metal ion concentrations, limiting the applicability of IPC as an indicator of the presence of metal inhibitor in a sample. Notably, ferrous ions were found to significantly decrease the extraction efficiency of the DNA‐IQ DNA extraction system. The amount of DNA degradation and inhibition in saliva samples caused by metal ions increased with a dilution of the sample, suggesting that the saliva matrix provides protection from metal ion effects.
Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence‐related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification ...(real‐time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an “inhibition study” and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in‐house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000‐fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR‐based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.
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