Prevalence of DNA from the driver, passengers and others within a car of an exclusive driver.

Cars are often sampled for DNA to help identify occupants and their possible location(s) within the car. While DNA from the frequent driver is likely to accumulate over time, DNA from previous and/or subsequent occupants, and those whose DNA has inadvertently been transferred to the car, may also contribute to any samples collected. This study investigates how much DNA resides on various sites within cars, and who might contribute to these samples. A total of 35-36 sites, internal and external, were targeted within four cars with sole long-term drivers. In addition to the car keys, sample sites included the exterior and interior door handles (driver and passenger sides), through to the internal compartments (driver side, middle area and front passengers' side). Reference samples were collected from the exclusive drivers, their co-resident partners and, where possible, recent passengers. The driver was always observed as a contributor in DNA profiles from the driver's side and, in most instances, was the sole, major or majority contributor to the profile. The driver was also observed as a major, majority or minor contributor at several sites on the passenger side. DNA of known recent passengers, close associates of the driver and unknown individuals was collected from many sites on both the driver and passenger sides. These findings may assist in sample targeting within cars and in the evaluation of DNA evidence when propositions relate to the activities performed.

Investigating the origins of eastern Polynesians using genome-wide data from the Leeward Society Isles.

The debate concerning the origin of the Polynesian speaking peoples has been recently reinvigorated by genetic evidence for secondary migrations to western Polynesia from the New Guinea region during the 2nd millennium BP. Using genome-wide autosomal data from the Leeward Society Islands, the ancient cultural hub of eastern Polynesia, we find that the inhabitants' genomes also demonstrate evidence of this episode of admixture, dating to 1,700-1,200 BP. This supports a late settlement chronology for eastern Polynesia, commencing ~1,000 BP, after the internal differentiation of Polynesian society. More than 70% of the autosomal ancestry of Leeward Society Islanders derives from Island Southeast Asia with the lowland populations of the Philippines as the single largest potential source. These long-distance migrants into Polynesia experienced additional admixture with northern Melanesians prior to the secondary migrations of the 2nd millennium BP. Moreover, the genetic diversity of mtDNA and Y chromosome lineages in the Leeward Society Islands is consistent with linguistic evidence for settlement of eastern Polynesia proceeding from the central northern Polynesian outliers in the Solomon Islands. These results stress the complex demographic history of the Leeward Society Islands and challenge phylogenetic models of cultural evolution predicated on eastern Polynesia being settled from Samoa.

Aboriginal Australian mitochondrial genome variation - an increased understanding of population antiquity and diversity.

Aboriginal Australians represent one of the oldest continuous cultures outside Africa, with evidence indicating that their ancestors arrived in the ancient landmass of Sahul (present-day New Guinea and Australia) ~55 thousand years ago. Genetic studies, though limited, have demonstrated both the uniqueness and antiquity of Aboriginal Australian genomes. We have further resolved known Aboriginal Australian mitochondrial haplogroups and discovered novel indigenous lineages by sequencing the mitogenomes of 127 contemporary Aboriginal Australians. In particular, the more common haplogroups observed in our dataset included M42a, M42c, S, P5 and P12, followed by rarer haplogroups M15, M16, N13, O, P3, P6 and P8. We propose some major phylogenetic rearrangements, such as in haplogroup P where we delinked P4a and P4b and redefined them as P4 (New Guinean) and P11 (Australian), respectively. Haplogroup P2b was identified as a novel clade potentially restricted to Torres Strait Islanders. Nearly all Aboriginal Australian mitochondrial haplogroups detected appear to be ancient, with no evidence of later introgression during the Holocene. Our findings greatly increase knowledge about the geographic distribution and phylogenetic structure of mitochondrial lineages that have survived in contemporary descendants of Australia's first settlers.

Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.

Deep Roots for Aboriginal Australian Y Chromosomes.

Australia was one of the earliest regions outside Africa to be colonized by fully modern humans, with archaeological evidence for human presence by 47,000 years ago (47 kya) widely accepted [1, 2]. However, the extent of subsequent human entry before the European colonial age is less clear. The dingo reached Australia about 4 kya, indirectly implying human contact, which some have linked to changes in language and stone tool technology to suggest substantial cultural changes at the same time [3]. Genetic data of two kinds have been proposed to support gene flow from the Indian subcontinent to Australia at this time, as well: first, signs of South Asian admixture in Aboriginal Australian genomes have been reported on the basis of genome-wide SNP data [4]; and second, a Y chromosome lineage designated haplogroup C(∗), present in both India and Australia, was estimated to have a most recent common ancestor around 5 kya and to have entered Australia from India [5]. Here, we sequence 13 Aboriginal Australian Y chromosomes to re-investigate their divergence times from Y chromosomes in other continents, including a comparison of Aboriginal Australian and South Asian haplogroup C chromosomes. We find divergence times dating back to ∼50 kya, thus excluding the Y chromosome as providing evidence for recent gene flow from India into Australia.

Preliminary investigation of differential tapelifting for sampling forensically relevant layered deposits.

The analysis of DNA mixtures can be problematic, especially when in trace quantities such as when a biological sample is deposited onto a substrate which contains background DNA (for example, in the case of touch DNA deposited onto a garment containing the wearer's DNA). We conducted a preliminary investigation into the possibility of removing such multi-donor deposits layer by layer using a differential tape-lifting method. Two types of tape were tested using two different numbers of applications for sampling layered deposits of touch DNA/touch DNA and touch DNA/saliva, both on the same polyester-cotton plain woven material. The data showed that there was no significant increase in the ratio of secondary to primary deposit when sampled in this manner, compared to direct extraction from cuttings of the touched fabric. A similar result was also obtained even when the deposits were on opposing surfaces of the fabric and the sampling was carried out on the secondary deposit side. These findings indicate that biological material, whether touch DNA or saliva, does not predominantly remain on the side of the fabric on which it is deposited (at least for plain-woven polyester-cotton). They also highlight the importance of considering substrate properties when making assumptions as to the resulting location of biological materials from a deposition event, and the necessity to conduct further research on the interactions between substrates and deposits.

FACS separation of non-compromised forensically relevant biological mixtures.

Although focusing attention on the statistical analysis of complex mixture profiles is important, the forensic science community will also benefit from directing research to improving the reduction of the incidence of mixtures before DNA extraction. This preliminary study analysed the use of fluorescence assisted cell sorting (FACS) for separation of cellular mixtures before DNA extraction, specifically mixtures of relatively fresh blood and saliva from two donors, prepared in 14 different mixture ratios. Improvements in the number of detectable alleles from the targeted cell type and overall profile quality were seen when compared to the results from unseparated samples. STRmix calculations revealed increases in likelihood ratios after separation, demonstrating the potential for higher probative values to be obtained from forensically relevant mixtures after subjecting them to FACS than from unsorted samples.

The use of forensic tests to distinguish blowfly artifacts from human blood, semen, and saliva.

This study investigated whether routinely used forensic tests can distinguish 3-day-old or 2-week-old fly artifacts, produced after feeding on human blood, semen, or saliva, from the biological fluid. Hemastix(®) , Hemident(™) , and Hemascein(™) were unable to distinguish blood from artifacts. Hemastix(®) returned false positives from negative controls. ABAcard(®) Hematrace(®) and Hexagon OBTI could distinguish blood from 3-day-old artifacts, but not 2-week-old artifacts. Phadebas(®) and SALIgAE(®) were unable to distinguish saliva from artifacts. RSID(™) -Saliva was able to distinguish saliva from 3-day-old artifacts, but not 2-week-old artifacts. Semen tests Seminal Acid Phosphatase, RSID(™) -Semen, and ABAcard(®) p30 were all able to distinguish semen from 3-day-old artifacts, but not 2-week-old artifacts. The tests investigated cannot be relied upon to distinguish artifacts from biological fluids. However, if an artifact is identified by its morphology, a positive result may indicate which biological fluid the fly consumed, and this knowledge may prove useful for investigators searching for DNA.

Evaluation of tapelifting as a collection method for touch DNA.

The use of tapelifting for collection of touch DNA from fabrics is routine in many jurisdictions. However, there is a paucity of data relating to the effectiveness of different types of tapes for tapelifting, the amount of tapelifting required to generate a useful profile, and whether or not tapelifting is more effective than swabbing from various substrates. This research investigates these questions by comparing two tapes of different adhesive strength currently used in forensic casework (Scotch Magic tape and Scenesafe FAST minitapes), for sampling from touch deposits on four different fabrics-cotton flannelette, cotton drill woven fabric, polyester/cotton plain woven fabric and polyester strapping. Touch DNA was deposited on four replicates of each substrate. Separate areas of each substrate replicate were sampled, either by taping with one of the two tapes or by wet/dry swabbing with cotton swabs. Tape was applied over the defined sampling area once or repeatedly for various numbers of applications. DNA was extracted, quantified and profiled from all tape and swab samples as well as the corresponding sampled substrates. Significantly more DNA was extracted, and a higher proportion of alleles detected, from Scenesafe FAST tape than from Scotch Magic tape. The amount of DNA and number of donor alleles detected generally increased as the tape was reapplied to the surface, although a threshold of collection was seen for both types of tape. For two out of four substrates, taping with Scenesafe FAST collected more DNA than swabbing and, for three substrates, generated a greater median number of donor alleles. There was no significant difference in numbers of alleles between swabbing and taping from flannelette. Based on these findings, it is recommended that a tape with stronger adhesion (such as Scenesafe FAST tapelifters) is generally preferable; that more than one application of tape is suggested (however, increasing the amount of times the area is sampled can diminish collection efficiency); and that there is an advantage using tapelifting rather than swabbing for fabrics unless, such as with flannelette, there are many loose fibres easily removed during the sampling process.

Persistence of DNA deposited by the original user on objects after subsequent use by a second person.

There is a paucity of data regarding the persistence of DNA from prior user of an object after, its use by another person. To acquire a greater understanding of persistence we performed controlled, experiments encompassing over 179 objects that had only been used by one individual for an extended, period before used by a 2nd person for various but known duration. Our findings show that the profile, percentage contribution of the 1st user relative to the 2nd user of an object declines in a linear manner, over time. The retrieval of the profile of the initial user of the object is dependent on the type of, substrate and use of the object. When considering a hard non-porous object the 1st user's profile, percentage contribution drops ∼50% immediately upon use by a 2nd person and drops to ∼15% after, 90 min. When considering a soft porous object the 1st wearer's profile contribution remains, higher than that of the 2nd wearer during the first 10h of wear by the 2nd wearer and still, accounts for ∼12% after 96 h. This substrate associated difference was also observed in an, assessment of a wide range of personal objects used by 2nd users for different durations. Particular, areas of certain objects were more likely to retain a greater proportion of the 1st user's DNA than other, areas. Alleles of unknown source were present on the majority of objects but rarely exceeded 10% of, the total profile. Greater knowledge of persistence will inform investigators regarding the likelihood of, detecting a profile of a particular individual based on the type of object and its history, and assist with, identifying the best areas of an object to target for DNA sampling.

Characterisation of novel and rare Y-chromosome short tandem repeat alleles in self-declared South Australian Aboriginal database.

Y-chromosome short tandem repeats (Y-STRs) are used in forensic science laboratories all over the world, as their application is wide and often vital in solving casework. Analysis of an in-house database of South Australian self-declared Aboriginal males held by Forensic Science South Australia (FSSA) using the Applied Biosystem's AmpFℓSTR® Yfiler™ PCR Amplification Kit revealed 43 variant Y-STR alleles at 6 of the 17 loci. All variant alleles were sequenced to determine the exact repeat structure for each. As a high level of admixture has previously been found within the SA Aboriginal database, samples were haplogrouped using Y-SNPs to determine their likely geographical origin. Although a number of variant alleles were associated with non-Aboriginal Y-haplogroups, a high frequency was observed within the Australian K-M9 lineage. Detailed knowledge of these variant alleles may have further application in the development of new DNA markers for identification purposes, and in population and evolutionary studies of Australian Aborigines.

The morphology of fecal and regurgitation artifacts deposited by the blow fly Lucilia cuprina fed a diet of human blood.

Fly feces and regurgitation deposits may be mistaken for bloodstain patterns at a crime scene, potentially compromising event reconstruction and/or misdirecting police resources. In some instances, these artifacts contain sufficient human biological material to generate a full DNA profile, sometimes 2 years after deposition. Clearly, it is important that investigators can make the distinction between artifacts and bloodstains. This study examined 6645 artifacts deposited on a smooth, nonporous surface after Lucilia cuprina were fed human blood. Artifacts were also compared with bloodstains on a variety of other surfaces. Both similarities and differences were found between artifacts and bloodstains, highlighting the need for an identification system to assist personnel with little training in bloodstain pattern analysis. The morphology of the artifacts has been described so that these deposits may be more clearly distinguished from bloodstains, targeted by crime scene personnel as potential sources of human DNA, and/or identified as potential evidence contaminants. Flowcharts have been devised to facilitate the analysis.

The influence of substrate on DNA transfer and extraction efficiency.

The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15µL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (p<0.05) differences also exist among the DNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (p<0.05) loss of DNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and concentration efficiencies among substrates is proposed.

An investigation of admixture in an Australian Aboriginal Y-chromosome STR database.

Y-chromosome specific STR profiling is increasingly used in forensic casework. However, the strong geographic clustering of Y haplogroups can lead to large differences in Y-STR haplotype frequencies between different ethnicities, which may have an impact on database composition in admixed populations. Aboriginal people have inhabited Australia for over 40,000 years and until ∼300 years ago they lived in almost complete isolation. Since the late 18th century Australia has experienced massive immigration, mainly from Europe, although in recent times from more widespread origins. This colonisation resulted in highly asymmetrical admixture between the immigrants and the indigenes. A State jurisdiction within Australia has created an Aboriginal Y-STR database in which assignment of ethnicity was by self-declaration. This criterion means that some males who identify culturally as members of a particular ethnic group may have a Y haplogroup characteristic of another ethnic group, as a result of admixture in their paternal line. As this may be frequent in Australia, an examination of the extent of genetic admixture within the database was performed. A Y haplogroup predictor program was first used to identify Y haplotypes that could be assigned to a European haplogroup. Of the 757 males (589 unique haplotypes), 445 (58.8%) were identified as European (354 haplotypes). The 312 non-assigned males (235 haplotypes) were then typed, in a hierarchical fashion, with a Y-SNP panel that detected the major Y haplogroups, C-S, as well as the Aboriginal subgroup of C, C4. Among these 96 males were found to have non-Aboriginal haplogroups. In total, ∼70% of Y chromosomes in the Aboriginal database could be classed as non-indigenous, with only 169 (129 unique haplotypes) or 22% of the total being associated with haplogroups denoting Aboriginal ancestry, C4 and K* or more correctly K(xL,M,N,O,P,Q,R,S). The relative frequencies of these indigenous haplogroups in South Australia (S.A.) were significantly different to those seen in samples from the Northern Territory and Western Australia. In S.A., K* (∼60%) has a much higher frequency than C4 (∼40%), and the subgroup of C4, C4(DYS390.1del), comprised only 17%. Clearly admixture in the paternal line is at high levels among males who identify themselves as Australian Aboriginals and this knowledge may have implications for the compilation and use of Y-STR databases in frequency estimates.

MtDNA SNP multiplexes for efficient inference of matrilineal genetic ancestry within Oceania.

Human mitochondrial DNA (mtDNA) is a convenient marker for tracing matrilineal bio-geographic ancestry and is widely applied in forensic, genealogical and anthropological studies. In forensic applications, DNA-based ancestry inference can be useful for finding unknown suspects by concentrating police investigations in cases where autosomal STR profiling was unable to provide a match, or can help provide clues in missing person identification. Although multiplexed mtDNA single nucleotide polymorphism (SNP) assays to infer matrilineal ancestry at a (near) continental level are already available, such tools are lacking for the Oceania region. Here, we have developed a hierarchical system of three SNaPshot multiplexes for genotyping 26 SNPs defining all major mtDNA haplogroups for Oceania (including Australia, Near Oceania and Remote Oceania). With this system, it was possible to conclusively assign 74% of Oceanian individuals to their Oceanian matrilineal ancestry in an established literature database (after correcting for obvious external admixture). Furthermore, in a set of 161 genotyped individuals collected in Australia, Papua New Guinea and Fiji, 87.6% were conclusively assigned an Oceanian matrilineal origin. For the remaining 12.4% of the genotyped samples either a Eurasian origin was detected indicating likely European admixture (1.9%), the identified haplogroups are shared between Oceania and S/SE-Asia (5%), or the SNPs applied did not allow a geographic inference to be assigned (5.6%). Sub-regional assignment within Oceania was possible for 32.9% of the individuals genotyped: 49.5% of Australians were assigned an Australian origin and 13.7% of the Papua New Guineans were assigned a Near Oceanian origin, although none of the Fijians could be assigned a specific Remote Oceanian origin. The low assignment rates of Near and Remote Oceania are explained by recent migrations from Asia via Near Oceania into Remote Oceania. Combining the mtDNA multiplexes for Oceania introduced here with those we developed earlier for all other continental regions, global matrilineal bio-geographic ancestry assignment from DNA is now achievable in a highly efficient way that is also suitable for applications with limited material such as forensic case work.

Increased amplification success from forensic samples with locked nucleic acids.

Inadequate sample quantities and qualities can commonly result in poor DNA amplification success rates for forensic case samples. In some instances, modifying the PCR protocol or components may assist profiling by overcoming inhibition, or reducing the threshold required for successful amplification and detection. Incorporation of locked nucleic acids (LNAs) into PCR primers has previously been shown to increase amplification success for a range of non-forensic sample types and applications. To investigate their use in a forensic context, the PCR primers for four commonly used STR loci have been redesigned to include LNA bases. The modified LNA primers provided significantly increased amplification success when compared to standard DNA primers, with both high-quality buccal samples and simulated forensic casework samples. Peak heights increased by as much as 5.75× for the singleplex amplifications. When incorporated into multiplexes, the LNA primers continued to outperform standard DNA primers, with increased ease of optimisation, and increased amplification success. The use of LNAs in PCR primers can greatly assist the profiling of a range of samples, and increase success rates from challenging forensic samples.

Forensic trace DNA: a review.

DNA analysis is frequently used to acquire information from biological material to aid enquiries associated with criminal offences, disaster victim identification and missing persons investigations. As the relevance and value of DNA profiling to forensic investigations has increased, so too has the desire to generate this information from smaller amounts of DNA. Trace DNA samples may be defined as any sample which falls below recommended thresholds at any stage of the analysis, from sample detection through to profile interpretation, and can not be defined by a precise picogram amount. Here we review aspects associated with the collection, DNA extraction, amplification, profiling and interpretation of trace DNA samples. Contamination and transfer issues are also briefly discussed within the context of trace DNA analysis. Whilst several methodological changes have facilitated profiling from trace samples in recent years it is also clear that many opportunities exist for further improvements.

Investigation of secondary DNA transfer of skin cells under controlled test conditions.

There is a paucity of data on the relative transfer rates of deposited biological substances which could assist evaluation of the probability of given crime scene scenarios, especially for those relating to objects originally touched by hand. This investigation examines factors that may influence the secondary transfer of DNA from this source, including the freshness of the deposit, the nature of the primary and secondary substrate and the manner of contact between the surfaces. The transfer rates showed that both the primary and secondary type of substrate and the manner of contact are important factors influencing transfer of skin cells, but, unlike other biological fluids, such as blood and saliva, the freshness of the deposit in most instances is not. Skin cells deposited on a non-porous primary substrate transferred more readily to subsequent substrates than those deposited on a porous substrate. Porous secondary substrates, however, facilitated transfer more readily than non-porous secondary substrates, from both porous and non-porous surfaces. Friction as the manner of contact significantly increased the rate of transfer. The findings of this study improve our general understanding of the transfer of DNA material contained in fingerprints that is left on a surface, and assist in the evaluation of the probability of secondary and further DNA transfer under specific conditions.

Genomic complexity of the Y-STR DYS19: inversions, deletions and founder lineages carrying duplications.

The Y-STR DYS19 is firmly established in the repertoire of Y-chromosomal markers used in forensic analysis yet is poorly understood at the molecular level, lying in a complex genomic environment and exhibiting null alleles, as well as duplications and occasional triplications in population samples. Here, we analyse three null alleles and 51 duplications and show that DYS19 can also be involved in inversion events, so that even its location within the short arm of the Y chromosome is uncertain. Deletion mapping in the three chromosomes carrying null alleles shows that their deletions are less than approximately 300 kb in size. Haplotypic analysis with binary markers shows that they belong to three different haplogroups and so represent independent events. In contrast, a collection of 51 DYS19 duplication chromosomes belong to only four haplogroups: two are singletons and may represent somatic mutation in lymphoblastoid cell lines, but two, in haplogroups G and C3c, represent founder lineages that have spread widely in Central Europe/West Asia and East Asia, respectively. Consideration of candidate mechanisms underlying both deletions and duplications provides no evidence for the involvement of non-allelic homologous recombination, and they are likely to represent sporadic events with low mutation rates. Understanding the basis and population distribution of these DYS19 alleles will aid in the utilisation and interpretation of profiles that contain them.

The Genographic Project public participation mitochondrial DNA database.

The Genographic Project is studying the genetic signatures of ancient human migrations and creating an open-source research database. It allows members of the public to participate in a real-time anthropological genetics study by submitting personal samples for analysis and donating the genetic results to the database. We report our experience from the first 18 months of public participation in the Genographic Project, during which we have created the largest standardized human mitochondrial DNA (mtDNA) database ever collected, comprising 78,590 genotypes. Here, we detail our genotyping and quality assurance protocols including direct sequencing of the mtDNA HVS-I, genotyping of 22 coding-region SNPs, and a series of computational quality checks based on phylogenetic principles. This database is very informative with respect to mtDNA phylogeny and mutational dynamics, and its size allows us to develop a nearest neighbor-based methodology for mtDNA haplogroup prediction based on HVS-I motifs that is superior to classic rule-based approaches. We make available to the scientific community and general public two new resources: a periodically updated database comprising all data donated by participants, and the nearest neighbor haplogroup prediction tool.