Ancestry and phenotype predictions from touch DNA using massively parallel sequencing.

Direct PCR can be used to successfully generate full STR profiles from DNA present on the surface of objects. STR profiles are only of use in cases where a potential donor profile is available for comparison, and DNA is of sufficient DNA quality and quantity to generate a reliable profile. Often, no donor information is available and only trace DNA is present on items. As a result, alternative techniques are required to generate genetic data that can provide investigative leads. Massively parallel sequencing (MPS) offers the ability to detect trace levels of DNA and improve DNA analysis success from touched items. Here, we present the first application of direct PCR coupled with MPS to generate forensic intelligence SNP data from latent DNA. The panels assessed are (1) the HIrisplex System that targets 24 SNPs to simultaneously predict hair and eye, and (2) the Precision ID Ancestry Panel that targets 165 autosomal SNPs indicative of biogeographic ancestry. For each panel, we analysed 60 touched samples across five individuals and four substrates (glass slide, fuse, zip-lock bag and wire) using Ion AmpliSeq Library Preparation Kit on the automated Ion Chef System and Ion Torrent PGM. We examine the SNP recovery, concordance with reference samples and the genotype reproducibility from different substrates and donors. The results demonstrate the application of this approach for obtaining informative genetic from trace amounts of DNA.

"Bottom-up" in situ proteomic differentiation of human and non-human haemoglobins for forensic purposes by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

RATIONALE: The detection and identification of human blood on crime-related items are of particular relevance to many investigations because shed blood can provide evidence of violent contact between individuals. However, for any detection and identification technique, specificity is a critical performance characteristic to assess; that is, whether the technique has the capability to differentiate between human blood (which usually is of relevance to a criminal investigation) and non-human blood (which usually would not be associated with a crime but may be detected incidentally). METHODS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approaches using "top-down" (detection of intact proteins) and "bottom-up" (detection of tryptic peptide markers) were used to detect and identify haemoglobin in blood from humans and from a range of Australian native mammals; the technique could be carried out directly on blood stains without the need to extract proteins (i.e., in situ measurement). Imaging of haemoglobin was achieved in bloodied fingermarks, including those that had been enhanced using two "industry standard" fingermark enhancement processes. RESULTS: Differentiation of intact haemoglobin proteins in human and non-human blood using "top-down" MALDI-TOF-MS was difficult. However, in situ "bottom-up" approaches using tandem mass spectrometry (MS/MS) and de novo sequencing of tryptic digest peptides allowed unambiguous differentiation. Imaging mass spectrometry of human haemoglobin, even when it was mixed with animal blood, was achieved in bloodied fingermarks that had been enhanced using two common processes (staining with Amido Black or dusted with magnetic powder) and "lifted" using adhesive tape. CONCLUSIONS: The MALDI-TOF-MS-based in situ "bottom-up" proteomic methodology described here shows great promise for the detection of human blood and even imaging of blood in bloodied fingermarks. The approach is sensitive, can differentiate between human blood and that from many animals (including several Australian native animals), and can be implemented after traditional crime scene fingermark enhancement techniques have been carried out.

Massively parallel sequencing is unlocking the potential of environmental trace evidence.

Massively parallel sequencing (MPS) has revolutionised the field of genomics enabling substantial advances in human DNA profiling. Further, the advent of MPS now allows biological signatures to be obtained from complex DNA mixtures and trace amounts of low biomass samples. Environmental samples serve as ideal forms of contact trace evidence as detection at a scene can establish a link between a suspect, location and victim. Many studies have applied MPS technology to characterise the biodiversity within high biomass environmental samples (such as soil and water) to address questions related to ecology, conservation, climate change and human health. However, translation of these tools to forensic science remains in its infancy, due in part to the merging of traditional forensic ecology practices with unfamiliar DNA technologies and complex datasets. In addition, people and objects also carry low biomass environmental signals which have recently been shown to reflect a specific individual or location. The sensitivity, and reducing cost, of MPS is now unlocking the power of both high and low biomass environmental DNA (eDNA) samples as useful sources of genetic information in forensic science. This paper discusses the potential of eDNA to forensic science by reviewing the most explored applications that are leading the integration of this technology into the field. We introduce novel areas of forensic ecology that could also benefit from these tools with a focus on linking a suspect to a scene or establishing provenance of an unknown sample and discuss the current limitations and validation recommendations to achieve translation of eDNA into casework.

Forensic validation of a SNP and INDEL panel for individualisation of timber from bigleaf maple (Acer macrophyllum Pursch).

Illegal logging is one of the largest illicit trades in the world, with high profits and generally low risks of detection and prosecution. Timber identification presents problems for law enforcement as traditionally used forensic methods such as wood anatomy and dendrochronology are often unable to confidently match wood evidence to the remains of illegally felled trees. Here we have developed and validated a set of genetic markers for individualisation in bigleaf maple (Acer macrophyllum), a high value timber species often felled illegally in the USA. Using 128 single nucleotide polymorphisms and three insertion/deletion markers developed through massively parallel sequencing, 394 individuals were genotyped on the MassARRAY® iPLEX™ platform (Agena Bio-science™, San Diego, USA) to produce a population reference database for the species. We demonstrate that the resulting DNA assay is reliable, species specific, effective at low DNA concentrations (<1 ng/µL) and suitable for application to timber samples. The PID for the most common profile, calculated using an overall dataset level FST-correction factor, was 1.785 × 10-25 and PID-SIB across all individuals (treated as a single population) was 2.496 × 10-22. The further development of forensic identification assays for timber species has the potential to deliver robust tools for improved detection and prosecution of illegal logging crimes as well as for the verification of legality in reputable supply chains.

Enhancement of fingermarks and visualizing DNA.

A novel method for detection and visualization of latent DNA using Diamond™ Nucleic Acid Dye (DD) staining has been developed. Applying DD to an object has the real potential to visualize DNA on a substrate from which a DNA profile can be generated, but it is important to determine whether this staining will adversely affect other forensic investigational techniques and vice versa. The aim of this study was to examine the interactions between staining a fingermark to detect DNA and then generate a DNA profile in combination with several standard latent fingermark enhancement methods. Six common fingerprint enhancements processes were chosen; (1) black powder, (2) black magnetic powder, (3) red magnetic powder, (4) white powder, (5) aluminum powder and (6) cyanoacrylate fuming. For all six methods, mark enhancement was carried out before DD staining and vice versa. DD is effective in detection of DNA in the presence of both aluminum and white finger mark powders and DD does not compromise the subsequent detection of ridge patterns if DD is applied first. Whilst magnetic powders could be used to successfully enhance latent fingermarks even after DD had been applied to them, latent DNA could not be observed in the marks irrespective of whether magnetic powder was applied before or after DD treatment. Magnetic powders did not adversely affect the profiling of DNA present in the marks. The application of DD to fingermarks did not adversely affect the enhancement of fingermarks using cyanoacrylate fuming. Whilst fluorescent particles resembling cells stained with DD were observed in marks either post-treated or pre-treated with cyanoacrylate vapor, DNA amplification and profiling was not successful. While it may be important in particular investigations to collect DNA profiles from latent fingermarks with continuous ridges and clear minutiae, the main utility of the technique described here would be in relation to investigations where enhancement has resulted in only partial or smudged marks. The results presented here indicate that if it is desirable to visualize latent DNA on an object but it is also planned to treat the object with cyanoacrylate vapor or magnetic powders then it is important to apply DD first, record the location of DNA and then apply the mark enhancement technique. For aluminum and white powder mark treatments such precautions are not important.

Visualising latent DNA on swabs.

Collection for touch DNA either at scenes or on items submitted to a forensic laboratory is based on assumptions as to where a person made direct contact. In many instances a swab may be applied to an area where no contact has been made. Many swabs may therefore be submitted for DNA profiling on which no DNA is present, resulting in the loss of both time and resources by analysing such swabs. This study has developed a simple, fast, DNA-staining and fluorescence microscopy-based screening method for swabs to indicate if there is any DNA from which to generate a profile. Ten different types of swabs were tested covering the major types used (foam, cotton and nylon). Each swab was treated by: no addition of dye or DNA, addition of dye only, addition of known DNA and addition of dye and DNA. The stain used was Diamond™ Nucleic Acid Dye (DD) and fluorescence microscopy was achieved with a digital microscope equipped with a blue LED light source (480nm) for excitation and an emission filter of 510nm. Two types of samples were tested, either buccal swabs or swabs collected from areas touched by volunteers and all analyses were performed in triplicate. The samples were collected and retained at room temperature with time intervals of 0 day, 7 days, 14 days, 21 days, and 28 days before detection using DD staining and fluorescence microscopy. Seven of the swab types used were found to be unsuitable due to the lack of any difference in the fluorescence detected when no DNA, or only the dye, or a combination of DNA and dye were added. Three swab types (black cotton swab, Ultrafine dental applicator, and Cylinder dental applicator) were found to be much more effective for collection of DNA. Further, stained cellular material retained its fluorescence for up to 4 weeks and swabs containing cellular material that had been stored for four weeks could be stained and visualised. Additionally, DD did not affect DNA profiling. This screening method has the potential to be a routine step in a forensic laboratory to save costs of processing samples where swabs are devoid of any DNA. This technique is rapid, easy, cheap, non-destructive and safe.

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

Detection and identification of cannabis by DNA.

The unambiguous identification of illicit substances, including Cannabis sativa, is a major concern of law enforcement agencies. Current methods of cannabis identification involve the use of techniques such as HPLC and GC to identify cannabinoids. A method for the identification of cannabis using DNA-specific primers has been developed and is described here. The nucleotide sequences between the trnL and trnF genes in the chloroplast of Cannabis sativa have been determined and Cannabis sativa-specific nucleotide sequences within the intergenic spacer between the trnL 3' exon and trnF gene identified. Primers, made to these sequences, have been tested on a range of different plant extracts but only give a PCR product in the presence of Cannabis sativa. The successful production of a PCR product using these primers identifies the presence of cannabis.

13 STR loci frequency data from a Scottish population.

A total of 13 STR loci were typed from over 200 Caucasians from the Strathclyde region in the west of Scotland.

Identification of members of the genera Panaeolus and Psilocybe by a DNA test. A preliminary test for hallucinogenic fungi.

Abuse of hallucinogens produced by the fungal genera Psilocybe and Panaeolus are a growing problem. Five species from each of the two genera were examined in this preliminary research and a method that will unambiguously identify fungal samples as being of one of these two genera has been developed. The method uses genus specific DNA sequences within the Internal Transcribed Spacer of the ribosomal gene complex. Amplification of a common DNA product and a genus specific product results in two identifiable products, which facilitates the unambiguous identification of material from these two fungi to generic level.

STR data for the GenePrint PowerPlex 1.2 system loci from three United Arab Emirates populations.

Allele frequencies for the eight STRs included in the GenePrint PowerPlex 1.2 kit were obtained from three population groups that are resident in the United Arab Emirates (UAE); 228 unrelated UAE individuals, 194 unrelated Indian individuals and 197 unrelated Pakistani individuals were analysed.

STR data for the SGM Plus loci from three Indonesian populations.

Allele frequencies for the 10 STRs included in the AmpFLSTR SGM Plus (Applied Biosytems) amplification kit were obtained from three populations in the Indonesian archipelago. Here, 173 unrelated Indonesian individuals were sampled, of which 44 were from the Island of Sulawesi, 44 from Sumatra and 85 from Java.

STR population data for 10 STR loci including the GenePrint PowerPlex 1.2 kit from El-Minia (Central Egypt).

Allele frequencies for 10 STRs including the GenePrint PowerPlex 1.2 loci and also D3S1358, HumvWA and HumFGA were obtained from a sample of unrelated individuals from El-Minia City.

Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population.

In order to demonstrate the sequence diversity of mitochondrial D-loop DNA in the Taiwanese Han population, we established a database of 155 unrelated individuals. For each individual, the complete 980bp DNA region from the 5' end of HVI to 3' end of HVII segment was sequenced. In these 155 sequence data, 149 different haplotypes were observed, amongst these haplotypes, 144 were unique, 4 were found in 2 individuals and 1 was found in 3 individuals. When compare to the Anderson sequence, 144 transitions, 24 transversions, 5 insertions and 5 deletions were found. Eight positions exhibited more than one polymorphic sequence, six exhibited two variants while two exhibited three variants. Over the 1024bp that was analysed, pairwise differences between the sequences were 11.35+/-3.53bp. The sequence and nucleotide diversity were 0.9994 and 0.0116, respectively. The probability of two individuals randomly matching over the entire control region was 0.007. The diversity in the mitochondrial D-loop indicates the value of this locus for casework within Taiwan.

The screening of 13 short tandem repeat loci in the Chinese population.

Population studies of 13 short tandem repeat (STR) loci were carried out on Chinese in Taiwan. The STR loci included HUMF13B, HUMF13A01, HUMFES/FPS, HUMFABP, HUMPLA2A1, HUMTPOX, HUMTH01, HUMVWFA31/A, HUMCSFIPO, HUMLPL, HUMGPP3A09, HUMCYAR04 and HUMCD4. DNA samples from 100 unrelated individuals were screened. The STR allele patterns were detected by the fluorescence detector of an automated DNA sequencer. Two PCR amplifications were performed for each STR locus in this study. The first PCR amplification strategy used 26 base pairs of the T7 sequence extension in the 5' end of the forward primer of each STR locus. The second PCR amplification used a dye-labeled T7 primer instead of the forward primer in the first PCR amplification, and the first PCR products as template to produce fluorescent dye-labeled PCR products. PCR products of different STR loci with overlapping allele sizes could be detected in the same lane of the polyacrylamide gel on an automated DNA sequencer using different colored dye-labeled T7 primers. There was no need to directly conjugate the fluorescent dye to individual STR primers. The PCR products were obtained using 2 ng of template DNA in 25 microliters of PCR reaction mixture. No deviations from the Hardy-Weinberg equilibrium were observed for the 13 STR loci. The distributions of these STR alleles were different from those of Caucasians or Blacks. The probability of matching from the combination of the 13 STR loci was 5.9 x 10(-10) for our Chinese population. However, HUMF13B, HUMLPL and HUMCD4 loci were not as highly polymorphic as observed in other populations.

Cytochrome b gene for species identification of the conservation animals.

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

HLA DQA1 genotyping of Chinese in Taiwan by PCR-RFLP.

Population data were established for the HLA DQA1 locus among 147 Chinese in Taiwan by the PCR-RFLP flowchart system. Twelve alleles could be differentiated in this study, although only nine alleles were found among the above samples. There were no discrepancies in the results obtained by PCR-RFLP when compared to the results obtained by the AmpliType HLA DQ alpha typing kit. Using the Chi-square test, significant differences were observed (p < 0.005) between this data and that for Japanese, African Americans and US Caucasians. The observed heterozygosity was 83% with a discrimination power of 0.93.

The detection and persistence of Cannabis sativa DNA on skin.

The presence of Cannabis sativa DNA was detected on the skin of persons who have recently handled both leaf and resinous material. The persistence of C. sativa DNA was examined on the skin. The subjects were asked to either repeatedly rub their hands on their trousers, place their hands repeatedly into their pockets or wash their hands in soap and water. After rubbing the hands on trousers or placing them in pockets C. sativa DNA could still be detected. No DNA could be detected after washing the hands.

Comparison of Cannabis sativa by random amplification of polymorphic DNA (RAPD) and HPLC of cannabinoids: a preliminary study.

Methods are described for the HPLC and genetic analysis of herbal Cannabis sativa. The latter method was applied to 17 plants grown simultaneously, at the same site. Sixteen of these samples were also compared using HPLC, which resulted in differentiation of the plants into 3 groups. Within two of these groups, the members could not be distinguished. By RAPD analysis, using certain combinations of primers and cladistic analysis, differentiation was possible between all but two of the plants. The use of the RAPD technique enables differentiation between samples that cannot be differentiated by HPLC analysis alone.

Forensic Science in Support of Wildlife Conservation Efforts - Genetic Approaches (Global Trends).

Wildlife forensic science is a relatively recent development to meet the increasing need of the criminal justice system where there are investigations in alleged transgressions of either international or national legislation. This application of science draws on conservation genetics and forensic geneticists from mainstream forensic science. This review is a broad overview of the history of forensic wildlife science and some of the recent developments in forensic wildlife genetics with the application of DNA developments to nonhuman samples encountered in a forensic science investigation. The review will move from methods to look at the entire genome, when there is no previous knowledge of the species studied, through methods of species identification, using DNA to determine a possible geographic origin, through to assigning samples to a particular individual or a close genetic relative of this individual. The transfer of research methods into the criminal justice system for the investigation of wildlife crimes has been largely successful as is illustrated in the review. The review concludes with comments on the need for standardization and regulation in wildlife forensic science.