Crime Scene Samples Research Articles

The Mysterious Murder of Christa Worthington

This lesson presents an interrupted case study based on the true story of the 2002 murder of Christa Worthington in Massachusetts. The case was developed for use in an undergraduate non-majors life science course, but would also be appropriate for a high school biology course or a forensic science course. During this lesson, students examine a crime scene diagram and discuss evidence collection. Students then conduct a hands-on activity extracting DNA from wheat germ to simulate how DNA would be isolated from crime scene samples. Lastly the students will analyze simulated DNA profiles produced using STRs, polymerase chain reaction, and gel electrophoresis to help match a crime scene sample to one of five suspects. The pros and cons surrounding the use of a DNA dragnet are also discussed.

RapidHIT for the purpose of stain analyses – An interrupted implementation

Rapid DNA instruments have in recent years been developed, enabling analysis of forensic samples with a minimum of human intervention. Initially intended for fast handling of reference samples, such as samples from suspects in booking suites, attention shifted to include crime scene samples. The aim of this study was to determine whether or not the RapidHIT System (IntegenX) is fit for crime scene samples. The first runs gave very poor results, which was found to be due to an incorrect firmware setting leading to no or just minute amounts of amplicons being injected for electrophoresis. After solving this problem, 28 full runs (seven samples each) applying NGM SElect Express were performed comprising various amounts of blood on cotton swabs. Six of the runs failed completely, four due to cartridge leakage and in two runs the PCR mix was not injected. For 155 samples with 1–5μL blood (volumes for which complete DNA profiles are expected), 119 samples (77%) gave complete DNA profiles. Among the most serious failures were incorrect allele calling and leakage of DNA extract or PCR product. Other general issues were failure to export results, anode motor breakdown and broken capillary array. Due to the encountered problems with software, hardware and cartridges, together with the low success rate, it was decided not to continue towards implementation of the RapidHIT System in casework.

The power of forensic DNA data bases in solving crime cases

Forensic DNA databases constitute an important investigative resource in contemporary criminal justice systems. The centralized and computerized storage of DNA profiles in a database enables the systematic comparison and automated matching of crime scene samples and individual profiles. Many countries operate forensic DNA-databases to identify owners of crime related stains. Using DNA to trace people who are suspected of committing a crime has been a major advance in policing. When DNA profiling is used wisely it can help to convict people who have committed serious crimes or exonerate people who are innocent. DNA database is a computer database containing records of DNA profiles. Usually there are two different sources of these DNA profiles: crime scene DNA samples and individuals’ DNA samples. The use of DNA databases in criminal investigations requires an individual’s identity to be revealed only if there is a match between their DNA profile and a crime scene DNA profile.We present two homicide cases where killers where identified by comparing the unknown STR’s profile found on ropes, which were used to tie the victims, and exhibit from the crime scene with the Macedonian forensic DNA database stored in Forensic department of MIA. Statistical analyses were performed using DNA View software.

Contamination incidents in the pre-analytical phase of forensic DNA analysis in Austria—Statistics of 17 years

Forensic DNA analyses have become more and more sensitive in the past years. With the ability to generate DNA profiles even from minute amounts of cellular material also the possibility to detect DNA on trace material that originates from persons not linked to the crime event, such as crime scene investigators, increases. The contamination of crime scene samples can lead to false positive results and misinterpretation that can cause deceptive investigations. In this work we continue a study of 2010 that compared the number of detected contamination incidents that were caused in the pre-analytical phase of forensic DNA analysis with the number of crime scene samples analyzed by our laboratory. Within the past 17 years we were able to detect a total of 347 contamination incidents caused by police officers in approximately 46,000 trace samples to their origin (0.75%). Additionally we demonstrate the usefulness of reference profile databases that contain DNA profiles of police officers to detect contamination incidents of trace material.

A Rapid, Confirmatory Test for Body Fluid Identification.

We have developed a technique that allows investigators to confirm the presence of blood, semen, and/or saliva in a crime scene sample. It is a confirmatory test where multiple samples can be processed in less than an hour, and it is potentially portable, permitting samples to be processed at the crime scene. Samples at a scene giving a positive result can be further processed while those failing to do so may be ignored. There is a large and growing backlog of DNA evidence in the USA, slowing down the criminal justice system. This backlog has continued to grow despite an increase in the ability to process evidence faster. This technique uses quantum dot molecular beacons to test for tissue-specific RNA species, identifying particular body fluids. We have demonstrated the tissue specificity of molecular beacons for blood, semen, and saliva.

Developmental validation of a 6-dye typing system with 27 loci and application in Han population of China

In this study, a novel 27-locus system (now known as the SureID PanGlobal system), including 24 autosomal STRs (D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D2S441, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33, D10S1248), two Y-chromosome markers (DYS391 and Y-indel) and the sex determining marker, Amelogenin was developed with six fluorescent dyes labeling. The included STR loci belonged to the core loci in the Combined DNA Index System (CODIS) and the European Standard Set (ESS) as well as some additional loci commonly used in commercial kits and national DNA databases. This paper describes the validation studies conducted with the SureID PanGlobal system using Applied Biosystems 3500 XL Genetic Analyzer for fragment length detection that included the analysis of the following parameters and aspects: PCR conditions, sensitivity, species specificity, inhibition, precision, stutter, DNA mixtures, and stability studies with crime scene samples. The studies demonstrated that the SureID PanGlobal system is reproducible, accurate, sensitive and robust for forensic application and databasing. Additionally, the whole cycling time of the system can finish within 65 minutes, which was developed specifically for rapid and reliable generation of DNA profiles obtained from blood, buccal swabs and forensic stains.

Report on the Identification of Different Y-STR Profiles between the Epithelial Cell Fraction (I) and the Sperm Fraction (II) of Samples from Sexual Assault Cases

최근 보급된 성폭력 응급 키트는 가해자의 타액이나 정액의 존재 여부를 확인하기 위하여 더 다양하고 세분화되었다. 이러한 이유로, 기존에 나타나지 않았던 Y-STR 프로필에 대한 여러 가지 특이한 사례들이 나타나고 있다. 여기에서는 국립과학수사연구원에서 감정했던 사례 중 Y-STR 프로필과 관련된 특이적인 두 사례에 대해 보고하고자 한다. 1번째 사례는, 정액반응 양성인 피해자의 생식기 채취 증거물(질 내용물, 자궁경부 닦은 면봉)에서 A (Autosomal)-STR 프로필은 피해자의 것만 검출되지만, 상피세포층(I)과 정자분획층(II)에서 서로 다른 남성의 Y-STR 프로필이 검출된 경우이다. 2번째 사례는, 정액반응 양성인 피해자의 생식기 채취 증거물(질 내용물)에서 남성①의 A-STR 프로필을 확보하였지만, 정자분획층(II)과 상피세포층(I)에서는 각각 남성① 및 남성②의 Y-STR 프로필이 검출된 경우이다. 2번째 사례에서, 남성①의 A-STR 프로필은 피의자의 A-STR 프로필과 일치하지 않았고, 오히려 상피세포 층(I)의 남성②의 Y-STR 프로필이 피의자의 Y-STR 프로필과 일치하였다. 이러한 사례들은 Y-STR 프로필과 관련된 감정을 더욱 신중하고 정확한 판단을 할 수 있도록 도움을 줄 것으로 사료된다.Recently supplied sexual assault evidence emergency kit is further subdivided and diversified to detect saliva and semen of suspect exactly. Therefore, some cases show an unique Y-STR profiling, which has not been reported. Here, we show two particular Y-STR typing cases performed in National Forensic Service. In the first case, only victim ’ s A (autosomal)-STR profile was detectable in the vaginal swab sample, which was positive for detection of semen. But, two different male s Y-STR profiles were obtained from the fraction of epithelial cells (I) and the fraction of sperm cells (II) of same evidence sample. In the second case, A-STR profile of one man (male ①) was obtained in the vaginal swab sample, which was positive for detection of semen. But, Y-STR profile of male① and Y-STR profile of another man (male②) were detected from FEC and FSC of same evidence sample, respectively. In this case, A-STR profile of male① did not match A-STR profile obtained from suspect. But, Y-STR profile of male① obatined from FEC was identical to suspect’s Y-STR profile. In conclusion, these cases will help to make more accurate decisions for the interpretation of Y-STR profiles obtained from crime scene samples.

YfilerⓇ Plus population samples and dilution series: stutters, analytic thresholds, and drop-out probabilities.

The Yfiler Ⓡ Plus Amplification Kit amplifies 27 Y chromosomal small tandem repeat (STR) markers. The kit has five-fluorescent dye chemistry and the improved PCR buffer system of modern STR kits. We validated the kit for accredited investigations of crime scene samples by a thorough study of kit dynamics and performance. We determined dye-dependent analytical thresholds by receiver operating characteristics (ROC) and made a customised artefact filter that includes theoretical known artefacts by use of previously analysed population samples. Dilution series of known male DNA and a selection of crime scene samples were analysed with the customised thresholds and artefact filters. The Yfiler Ⓡ Plus Amplification Kit was sensitive giving full profiles down to 70 pg of male DNA. The balances between the fluorescent dyes as well as between loci were very good. The kit was able to produce full Y-STR profiles from crime scene samples containing small amounts of male DNA and large amounts of female DNA (although unspecific reactions were evident for very unbalanced mixtures). A decrease in the drop-out rate was found for both the dilution series and population samples, as well as a small increase in the drop-in rate for population samples, using the customised threshold and artefact filters compared to company-provided thresholds and artefact filters. The additional drop-ins were all of a nature that would be detected by inspection of the results. For the crime scene samples, large amounts of female DNA complicated the analysis by causing drop-ins of characteristic female DNA artefacts. Even though the customised analytical threshold in combination with the custom-made artefact filters gave more alleles, crime scene samples still needed special attention from the forensic geneticist.

Rapid microfluidic analysis of a Y-STR multiplex for screening of forensic samples.

In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80s. The entire procedure takes less than 25min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework. Graphical abstract ᅟ.

Encoding of low-quality DNA profiles as genotype probability matrices for improved profile comparisons, relatedness evaluation and database searches

Many DNA profiles recovered from crime scene samples are of a quality that does not allow them to be searched against, nor entered into, databases. We propose a method for the comparison of profiles arising from two DNA samples, one or both of which can have multiple donors and be affected by low DNA template or degraded DNA. We compute likelihood ratios to evaluate the hypothesis that the two samples have a common DNA donor, and hypotheses specifying the relatedness of two donors. Our method uses a probability distribution for the genotype of the donor of interest in each sample. This distribution can be obtained from a statistical model, or we can exploit the ability of trained human experts to assess genotype probabilities, thus extracting much information that would be discarded by standard interpretation rules. Our method is compatible with established methods in simple settings, but is more widely applicable and can make better use of information than many current methods for the analysis of mixed-source, low-template DNA profiles. It can accommodate uncertainty arising from relatedness instead of or in addition to uncertainty arising from noisy genotyping. We describe a computer program GPMDNA, available under an open source licence, to calculate LRs using the method presented in this paper.

Developmental Validation of a novel 5 dye Y-STR System comprising the 27 YfilerPlus loci.

In this study, a new STRtyper-27 system, including the same Yfiler Plus loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS449, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627 and DYF387S1a/b), was established using a set of 5 fluorescent dye labels. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house for this multiplex system. This paper describes the validation studies conducted with the STRtyper-27Y system using a 3130XL genetic analyzer for fragment length detection that included the analysis of the following parameters and aspects: sensitivity, species specificity, inhibition, haplotype concordance, precision, stutter, DNA mixtures, and stability studies with crime scene samples. The studies demonstrated, that the STRtyper-27Y system provided equivalent overall performance comparable to the latest Yfiler Plus kit, but with enhanced compatibility in terms of instrument platforms and software allowing forensic laboratories to conduct its forensic application and evaluate its performance, all in their own 5 dye Y-STR chemistry system /environment without software or instrument upgrades.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood

The ability to predict the type of tissues or cells from molecular profiles of crime scene samples has important practical implications in forensics. A previously reported multiplex assay using DNA methylation markers could only discriminate between 4 types of body fluids: blood, saliva, semen, and the body fluid which originates from female reproductive organ. In the present study, we selected 15 menstrual blood-specific CpG marker candidates based on analysis of 12 genome-wide DNA methylation profiles of vaginal fluid and menstrual blood. The menstrual blood-specificity of the candidate markers was confirmed by comparison with HumanMethylation450 BeadChip array data obtained for 58 samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid, and 19 skin epidermis samples. Among 15CpG marker candidates, 3 were located in the promoter region of the SLC26A10 gene, and 2 of them (cg09696411 and cg18069290) showed high menstrual blood specificity. DNA methylation at the 2CpG markers was further tested by targeted bisulfite sequencing of 461 additional samples including 49 blood, 52 saliva, 34 semen, 125 vaginal fluid, and 201 menstrual blood. Because the 2 markers showed menstrual blood-specific methylation patterns, we modified our previous multiplex methylation SNaPshot reaction to include these 2 markers. In addition, a blood marker cg01543184 with cross reactivity to semen was replaced with cg08792630, and a semen-specific unmethylation marker cg17621389 was removed. The resultant multiplex methylation SNaPshot allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood using the 9CpG markers which show a methylation signal only in the target body fluids. Because of the complexity in cell composition, menstrual bloods produced DNA methylation profiles that vary with menstrual cycle and sample collection methods, which are expected to provide more insight into forensic menstrual blood test. Moreover, because the developed multiplex methylation SNaPshot reaction includes the 4CpG markers of which specificities have been confirmed by multiple studies, it will facilitate confirmatory tests for body fluids that are frequently observed in forensic casework.

High-throughput DNA extraction of forensic adhesive tapes

Tape-lifting has since its introduction in the early 2000′s become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the “stickiness”, rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples.

Contamination when collecting trace evidence—An issue more relevant than ever?

With the development of more sensitive DNA amplification and STR typing kits, the chance of detecting minute contamination of trace material increases. At the same time, possible ways of detecting unwanted contamination of crime scene samples were also enhanced. To estimate the contamination rate of biological traces nationwide, we continued a study from 2010 that compares the number of analyzed crime scene samples with the number of detected contamination incidents within our laboratory. The results of this study illustrate the importance of reference profiles, especially of crime scene investigators, and the advantage of database assisted profile alignment in cases of mixtures.

Performing body fluid identification with microRNAs using capillary electrophoresis

MicroRNAs have a potential to be ideal forensic markers due to their small size (∼22nt), high abundance per cell, and sensitive and specific detection. Thousands of microRNAs are present in biological material and they are suitable for body fluid identification (BFID). Their advantageous properties increase the chances of successful analysis from challenged crime scene samples. In addition, it has been demonstrated that informative microRNA expression levels can be obtained from common DNA extracts. Following an earlier pilot project on a single stream process with the integration of microRNA analysis into a DNA profiling multiplex, progress on this line of research is now presented. A panel of 8 microRNAs (hsa-miR-10a, -16a, -135a, -142, -203a, -205, -451a and -1260b) has been identified to allow differentiation between blood, saliva, semen and vaginal material. Here the analysis of the BFID markers using capillary electrophoresis (CE) on ABI’s 3130 genetic analyser is presented. The markers are reverse transcribed using a multiplex stem-loop reverse transcription, followed by PCR with ROX-labelled universal reverse primer for the detection of cDNA.It is shown that – after careful optimization – BFID microRNA analysis using CE has similar discriminatory power as using qPCR in singleplex reactions and is therefore a viable technique for BFID. Multiplexing these markers is a next step that can result in a single test for BFID with the advantageous properties of microRNAs.

SNPforID 52-plex in casework samples: “Cracking” bones and other difficult samples

Casework samples can present difficulties to forensic scientists in criminal and identification investigations. Some challenging samples like bones, teeth and crime scene samples, often contain low DNA quantity which can even be degraded. In these cases, obtained STR profiles are many times incomplete or even null. This is partly due to relative bigger size of commonly used STRs in forensic analysis. In order to bypass this problem, other strategies of analysis have been developed in the past based on mini-STRs and biallelic markers, such as Indels and SNPs. Although each marker type has its advantages and disadvantages, SNPs benefit from the fact of having smaller amplification products and its analysis can be realized analyzing simultaneously a great number of loci using large multiplexes. One of such multiplex is SNPforID 52-plex which analyzes 52 loci, providing good results, as reported by some authors. Taking this in consideration, we compared the amplification success of 53 real casework samples from our casuistic consisting of bones, teeth and other samples using the 52-plex and the Identifier® Plus kit. Mean amplification success rate by loci was of 73% and 43% respectively and 16 out of 36 samples in which STR profiles were not obtained or in which these were poor, generated complete or almost complete SNP profiles. We conclude that the 52-plex can be a valuable tool in the analysis of different types of challenging forensic samples when STRs fail to provide necessary genetic information for identification.

Re-evaluation of the Seratec® PSA Semiquant test: comparison of kit provided buffer with phosphate buffer saline

The Seratec® PSA Semiquant test has been utilized in the forensic community to help identify seminal fluid on crime scene samples. The value of the test is dependent on its ability to accurately identify a prostate specific antigen (PSA) in various samples. Several non-biological substances that may be found on oral and vaginal samples in forensic casework were tested in this study to see if they would give false positive results using the Seratec® PSA Semiquant kit with two different buffers. Results indicate Red Bull® Energy Drink, Rock Star® Sugar Free Energy Supplement, and Summer's Eve® Douche Medicated as non-biological substances that gave false positive results when extracted with Phosphate Buffer Saline (PBS) on the test strips. False positive results were also observed on oral swabs collected from individuals who ingested Red Bull® Energy Drink. The false positive results can be attributed in part to a drop in pH of the PBS buffer. The buffer provided with the Seratec® PSA Semiquant kit was also tested and was found to prevent these false positive results from occurring. This Seratec® buffer was seen to effectively maintain a pH of 8.5 for all samples tested and was just as sensitive as the PBS buffer. This study illustrates the need for laboratories that use the Seratec® PSA Semiquant kit to re-evaluate the buffer they are using to extract case samples.

Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM

DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine® (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1ng of genomic DNA and mRNA analysis from 10ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material.

Predicting biogeographical ancestry in admixed individuals – values and limitations of using uniparental and autosomal markers

When microsatellite profiles generated from crime scene samples do not match a known person, or eye-witness information is unreliable, highly informative uniparental and autosomal markers can help unveil biogeographical ancestry. However, as genetic admixture is becoming increasingly common in cosmopolitan societies, concern arises with their accuracy and suitability when dealing with samples from admixed individuals. Here we assess the ability to detect biogeographical ancestry in 85 individuals from self-declared Asian and European admixed families using a set of uniparental (Y and mitochondrial DNA) and autosomal single nucleotide polymorphisms, specifically selected to distinguish between these two biogeographical ancestries. Haplogroups and autosomal genotypes were investigated using STRUCTURE to detect levels of admixture. All haplogroups were characteristic of self-declared populations of origin. Overall, the autosomal markers inferred biogeographical ancestry more accurately in admixed individuals, showing no significant differences between observed and expected contribution from each population studied according to level of admixture, although some outliers were observed. We suggest a panel of highly informative autosomal and uniparental markers should be employed to infer biogeographical ancestry of an individual to help detect admixed ancestries.

Predicting the origin of soil evidence: High throughput eukaryote sequencing and MIR spectroscopy applied to a crime scene scenario

Soil can serve as powerful trace evidence in forensic casework, because it is highly individualistic and can be characterised using a number of techniques. Complex soil matrixes can support a vast number of organisms that can provide a site-specific signal for use in forensic soil discrimination. Previous DNA fingerprinting techniques rely on variations in fragment length to distinguish between soil profiles and focus solely on microbial communities. However, the recent development of high throughput sequencing (HTS) has the potential to provide a more detailed picture of the soil community by accessing non-culturable microorganisms and by identifying specific bacteria, fungi, and plants within soil. To demonstrate the application of HTS to forensic soil analysis, 18S ribosomal RNA profiles of six forensic mock crime scene samples were compared to those collected from seven reference locations across South Australia. Our results demonstrate the utility of non-bacterial DNA to discriminate between different sites, and were able to link a soil to a particular location. In addition, HTS complemented traditional Mid Infrared (MIR) spectroscopy soil profiling, but was able to provide statistically stronger discriminatory power at a finer scale. Through the design of an experimental case scenario, we highlight the considerations and potential limitations of this method in forensic casework. We show that HTS analysis of soil eukaryotes was robust to environmental variation, e.g. rainfall and temperature, transfer effects, storage effects and spatial variation. In addition, this study utilises novel analytical methodologies to interpret results for investigative purposes and provides prediction statistics to support soil DNA analysis for evidential stages of a case.