Next‐Generation Sequencing of the Bacterial 16S rRNA Gene for Forensic Soil Comparison: A Feasibility Study

Soil has the potential to be valuable forensic evidence linking a person or item to a crime scene; however, there is no established soil individualization technique. In this study, the utility of soil bacterial profiling via next‐generation sequencing of the 16S rRNA gene was examined for associating soils with their place of origin. Soil samples were collected from ten diverse and nine similar habitats over time, and within three habitats at various horizontal and vertical distances. Bacterial profiles were analyzed using four methods: abundance charts and nonmetric multidimensional scaling provided simplification and visualization of the massive datasets, potentially aiding in expert testimony, while analysis of similarities and k‐nearest neighbor offered objective statistical comparisons. The vast majority of soil bacterial profiles (95.4%) were classified to their location of origin, highlighting the potential of bacterial profiling via next‐generation sequencing for the forensic analysis of soil samples.     

Time Radically Alters Ex Situ Evidentiary Soil 16S Bacterial Profiles Produced Via Next‐Generation Sequencing,,

Previous research has revealed the potential of soil bacterial profiling for forensic purposes; however, investigators have not thoroughly examined fluctuations in microbial profiles from soil aged on evidence. In this research, soils collected from multiple habitats were placed on evidence items and sampled over time, and then bacterial profiles were generated via next‐generation sequencing of the 16S rRNA locus. Bacterial abundance charts and nonmetric multidimensional scaling plots provided visual representation of bacterial profiles temporally, while supervised classification was used to statistically associate evidence to a source. The ex situ evidence soils displayed specific, consistent taxonomic changes as they aged, resulting in their drift in multidimensional space, but never toward a different habitat. Ninety‐five percent of the 364 evidentiary profiles statistically classified to the correct habitat, with misclassification generally stemming from evidence type and increased age. Ultimately, understanding bacterial changes that occur temporally in ex situ soils should enhance their use in forensic investigations.     

Identifying background microbiomes in an evidence recovery laboratory: A preliminary study

16S rRNA profiling of bacterial communities may have forensic utility in the identification or association of individuals involved with criminal activities. Microbial profiling of evidence may, in the future, be performed within environments currently utilised for human DNA recovery, such as a forensic biology laboratory. It would be important to establish the background microbiome of such an environment to determine the potential presence of human or environmental microbial signatures to assist forensic scientists in the appropriate interpretation of target microbial communities. This study sampled various surfaces of an Evidence Recovery Laboratory (ERL) on three occasions including (a) before a monthly deep-clean, (b) immediately following the deep-clean, and (c) immediately after the laboratory’s use by a single participant for the purposes of routine item examinations. Microbial profiles were also generated for the involved participant and researcher for comparison purposes. Additionally, human nuclear DNA was profiled for each of the samples collected, using standard forensic profiling techniques, to provide a prospective link to the presence or absence of a background microbial signature within the ERL after its use. Taxonomic distributions across ERL samples revealed no consistent signature of any of the items sampled over time, however, major phyla noted within all ERL samples across the three timepoints were consistent with those found in human skin microbiomes. PCoA plots based on the Unweighted Unifrac metric revealed some clustering between participant microbial reference samples and surfaces of the ERL after use, suggesting that despite a lack of direct contact, and adherence to standard operating procedures (SOPs) suitable for human DNA recovery, microbiomes may be deposited into a forensic setting over time. The reference samples collected from the involved participant and researcher generated full STR profiles. Human DNA was observed to varying degrees in samples taken from the ERL across each of the sampling timepoints. There was no correlation observed between samples that contained or did not contain detectable quantities of human nuclear DNA and microbial profile outputs.     

Analysis of forensic soil samples via high-performance liquid chromatography and ion chromatography

Traditional forensic soil comparisons are performed via physical and/or chemical examinations of color, texture, and mineral content, leaving any organic- or water-soluble fractions unexamined. This study uses high-performance liquid chromatography (HPLC) and ion chromatography (IC) to assess the qualitative and quantitative variation in these fractions of soil. Soil samples (n=120) were collected over the course of 3 weeks from urban, suburban, and rural locations in and around Lansing, MI. Additional samples from six of these locations (two urban, two suburban, and two rural) were collected once a week for 10 weeks for temporal analysis. Nine additional samples, equally spaced over a 1 m(2) grid, from these same six locations were collected for spatial analyses. Qualitative and quantitative analysis of the resultant chromatograms separated the 120 samples into 10 groups by HPLC and 23 groups by IC. This study shows that using HPLC and IC to analyze the organic- and water-soluble fractions of soil can successfully discriminate samples. Quantitative analysis of the results eliminates some false inclusions by providing further differentiation of samples. The results of this study indicate that adding HPLC and IC analyses to traditional forensic soil analysis schemes can improve overall sample differentiation. The methods used in this study were also able to detect both qualitative and quantitative variations in soil over a relatively small geographic area. This demonstration of soil heterogeneity underscores the importance of the collection of a representative known sample population when assessing a forensic soil comparison. Significant temporal variation was also demonstrated over the course of 10 weeks of sampling; however, samples were found to be consistent over shorter periods of time. Baseline levels of inorganic anions were determined via IC; these levels may be useful in assessing the significance of anions detected in soil from cases involving low explosives.     

土壤细菌群体多样性的T-RFLP分析应用探讨

目的探讨土壤细菌群体多样性的末端限制性片段长度多态性（terminal restriction fragment length polymorphism，T-RFLP）分析在法庭科学应用的相关问题。方法依据不同土壤中的细菌群体存在多样性和差异，联合利用细菌16SrDNA序列、末端限制性片段长度多态性（terminal restriction fragment length polymorphism，T-RFLP）方法对5个来源不同的土壤样品和4个同一来源土壤样品的细菌群体多样性进行比较分析，计算土壤样品间的相似系数。结果不同来源的5个土壤样品间相似系数，最大者为0．44，最小为0．3；同一来源的4个土壤样品相似系数，最大为0．87，最小为0．76。结论不同来源土壤的细菌群体多样性存在差异。     

Bacterial Profiling of Soil Using Genus‐Specific Markers and Multidimensional Scaling*

Abstract: Forensic identification of soil based on microbial DNA fingerprinting has met with mixed success, with research efforts rarely considering temporal variability or local heterogeneity in soil’s microbial makeup. In the research presented, the nitrogen fixing bacteria rhizobia were specifically examined. Soils were collected monthly from five habitats for 1 year, and quarterly in each cardinal direction from the main collection site. When all habitats were compared simultaneously using Terminal Restriction Fragment Length Polymorphism analysis of the rhizobial recA gene and multidimensional scaling, only two were differentiated over a year’s time, however pairwise comparisons allowed four of five soils to be effectively differentiated. Adding in 10‐foot distant soils as “questioned” samples correctly grouped them in 40–70% of cases, depending on restriction enzyme used. The results indicate that the technique has potential for forensic soil identification, although extensive anthropogenic manipulation of a soil makes such identification much more tentative.     

Bacterial Profiling of Soil For Forensic Investigations: Consideration of Ex Situ Changes in Questioned and Known Soil Samples

Soil, being diverse and ubiquitous, can potentially link a suspect or victim to a crime scene. Recently scientists have examined the microbial makeup of soil for determining its origin, and differentiating soil samples is well-established. However, when soil is transferred to evidence its microbial makeup may change over time, leading to false exclusions. In this research, “known” soils from diverse habitats were stored under controlled conditions, while evidence soils were aged on mock evidence. Limited quantities of soil were also assayed. Bacterial profiles were produced using next-generation sequencing of the 16S rRNA gene. Overall, known soils stored open at room temperature were more similar to evidence soils over time than were known soils stored bagged and/or frozen. Evidence soils, even as little as 1 mg, associated with the correct habitat 99% of the time, accentuating the importance of considering ex situ microbial changes in soil for its successful use as forensic evidence.     

Assessing the potential of bacterial DNA profiling for forensic soil comparisons

A pilot study was undertaken to evaluate DNA profiling of the bacterial community in soil as an alternative to geological methods for forensic soil comparisons. Soil samples from three different ecosystems were compared, and the variation within and between ecologically different sites was determined by using terminal restriction fragment (TRF) analysis of 16S ribosomal DNA. Comparison of TRF profiles revealed that samples from within a specific ecosystem (e.g., a field) showed a significantly higher similarity to each other than to those from another ecosystem (e.g., a forest). In addition, some profile features were unique to specific ecosystems. These features may allow the determination of characteristic profiles that will facilitate identification of ecologically different sites, so that a given sample collected from a suspect could be identified as originating from, for example, a field, rather than a forest. The implications of these preliminary findings for forensic investigations are discussed.     

Soil sample metagenome NGS data management for forensic investigation

Soil analysis is a valuable resource in forensic investigation. Classical forensic soil analysis involves examination of its physical characteristics and chemical composition, such as soil type, colour, particle size, shape, pH, elemental, mineral and organic content. However the limited variability of these parameters is not always allowing adequate discrimination between soil samples. As soil supports extreme diversity of microorganisms and eukaryotic communities, microbiological approaches have been proposed. Several molecular approaches for microbial DNA profiling are available; however there is a lack of published data of implementation of the next generation sequencing (NGS) approaches for forensic soil analysis.The aim of the current study was elaboration of criteria for soil metagenome data management and database searching. We used our previously sequenced collection of 11 samples collected from different environments (forests, fields, grasslands, urban park) with different flora. The single sample collection includes 9 soil samples per one sampling area (30 m × 30 m) spaced by 15 m. In the current study we concentrated mainly on 18S rRNA gene V2-V3 region for fungi however SSU rRNA region for arbuscular mycorrhizal (AMF) fungi and V2-V3 hypervariable region of 16S rRNA gene for bacterial communities were taken into account. The sequencing was performed by Roche/454 platform. For data analysis OTU based approach on mothur software and NCBI BLASTN search were used. NCBI BLASTN analysis revealed altogether 2983 AMF matches and 8997 18S matches as well as 25477 OTUs (16S) were determined. Several data filtration approaches were used for data management. We found that 18S marker results could be used to create and run a filtered database that is computationally much more efficient and flexible. Our results have broad impact; however more samples have to be analysed, additional studies performed and cooperation between soil scientists and forensic scientists is required to be able to implement these novel techniques into the routine forensic practice.     

Microbial metagenome profiling using amplicon length heterogeneity-polymerase chain reaction proves more effective than elemental analysis in discriminating soil specimens

The combination of soil's ubiquity and its intrinsic abiotic and biotic information can contribute greatly to the forensic field. Although there are physical and chemical characterization methods of soil comparison for forensic purposes, these require a level of expertise not always encountered in crime laboratories. We hypothesized that soil microbial community profiling could be used to discriminate between soil types by providing biological fingerprints that confer uniqueness. Three of the six Miami-Dade soil types were randomly selected and sampled. We compared the microbial metagenome profiles generated using amplicon length heterogeneity-polymerase chain reaction analysis of the 16S rRNA genes with inductively coupled plasma optical emission spectroscopy analysis of 13 elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Si, and Zn) that are commonly encountered in soils. Bray-Curtis similarity index and analysis of similarity were performed on all data to establish differences within sites, among sites, and across two seasons. These data matrices were used to group samples that shared similar community patterns using nonmetric multidimensional scaling analysis. We concluded that while chemical characterization could provide some differentiation between soils, microbial metagenome profiling was better able to discriminate between the soil types and had a high degree of reproducibility, therefore proving to be a potential tool for forensic soil comparisons.     

Potential carcass enrichment of the University of Tennessee Anthropology Research Facility: A baseline survey of edaphic features

The University of Tennessee Anthropology Research Facility (ARF) is known for its unique contribution to forensic science as a site of human decomposition research. Studies conducted at ARF are integral in our understanding of the processes of human decomposition. As such, the authors are interested in the long-term effects of continuous human decomposition on the soil environment. Soil samples collected from within and outside the ARF were evaluated for moisture content, pH, organic content, total carbon and nitrogen content, and biomass by lipid-bound phosphorus, and total extracted DNA. Analyses revealed no significant differences (p<0.05) among the sampled areas within the facility, and yet demonstrated a possible trend toward increased levels of total N, Lipid-P, and water, suggesting an influx of high-quality nutrients into the ARF soil. Furthermore, elevated pH readings, presumably resulting from ammonification of the soil, were observed in areas of high decomposition. The negative control samples proved significantly different from nearly all samples collected within the facility, the exceptions being total carbon content and extractable DNA. These findings indicate that while landscape samples inside may be similar to themselves, they are dissimilar to those taken in a similar temperate forest biome with no recorded history of human decomposition.     

Discrimination of Soils at Regional and Local Levels Using Bacterial and Fungal T‐RFLP Profiling*

Abstract: DNA profiling of microbial communities has been proposed as a tool for forensic comparison of soils, but its potential to discriminate between soils from similar land use and/or geographic location has been largely unexplored. We tested the ability of terminal restriction fragment length polymorphism (T‐RFLP) to discriminate between soils from 10 sites within the Greater Wellington region, New Zealand, based on their bacterial and fungal DNA profiles. Significant differences in bacterial and fungal communities between soils collected from all but one pair of sites were demonstrated. In some instances, specific terminal restriction fragments were associated with particular sites. Patch discrimination was evident within several sites, which could prove useful for site‐specific matching (e.g., matching shoe/car tire print to an object). These results support the need for further understanding of the spatial distribution of soil microbial communities before DNA profiling of soil microbial communities can be applied to the forensic context.     

SEM-EDS analysis and discrimination of forensic soil

Soils vary among different areas, and have some characteristics because of the natural effects and transfers made by human and other living beings in time. So that forensic examination of soil is not only concerned with the analysis of naturally occurring rocks, minerals, vegetation, and animal matter. It also includes the detection of such manufactured materials such as ions from synthetic fertilizers and from different environments (e.g., nitrate, phosphate, and sulfate) as environmental artifacts (e.g., lead or objects as glass, paint chips, asphalt, brick fragments, and cinders) whose presence may impart soil with characteristics that will make it unique to a particular location. Many screening and analytical methods have been applied for determining the characteristics which differentiate and discriminate the forensic soil samples but none of them easily standardized. Some of the methods that applied in forensic laboratories in forensic soil discrimination are the color comparison of the normal air-dried (dehumidified) and overheated soil samples, macroscopic observation, and low-power stereo-microscopic observation, determination of anionic composition by capillary electrophoresis (CE), and the elemental composition by scanning electron microscope (SEM)-energy dispersive X-ray spectrometer (EDS) and other high sensitivity techniques. The objective of this study was to show the effect of the application of 9 tonnes/cm2 pressure on the elemental compositions obtained by SEM-EDS technique and comparing the discrimination power of the pressed-homogenized and not homogenized forensic soil samples. For this purpose soil samples from 17 different locations of Istanbul were collected. Aliquots of the well mixed samples were dried in an oven at 110-120 degrees C and sieved by using 0.5 mm sieve and then the undersieve fraction(<0.5 mm) of these samples put on an adhesive tape placed on a stub. About 100-150 mg aliquots of dried, sieved samples were pressed under 9 tonnes/cm2 pressure by KBr disk preparation apparatus of an infrared spectrophotometer. Surfaces of the randomized particles and the pressed disks of the soil samples were scanned and the elemental compositions were determined with scanning electron microscope JEO-JSM-5600 equipped with an energy dispersive X-ray spectrometer OXFORD Link-ISIS-300. The samples from top of the sieves were examined with stereo-microscope equipped with JVC-TK-128DE color video camera and JVC-GV-PT2, digital video printer. Natural and artificial materials that have characteristic features were identified. Then for additional confirmation all soil samples were dried at 120 degrees C and over 780 degrees C and their colors compared. We concluded that pressing the whole sieved soil samples under 9 tonnes/cm2 pressure results in smashing over the harder particles into the softer matrix and results in homogenization of the soil sample. The elemental compositions of these samples obtained by SEM-EDS with 10-fold less standard deviation (S.D.) values and so that with more reproducibility and discrimination power.     

The Potential of High-throughput Metagenomic Sequencing of Aquatic Bacterial Communities to Estimate the Postmortem Submersion Interval

Human remains can be discovered in freshwater or marine ecosystems, circumstances where insects and other invertebrates have infrequently been used for understanding the time of postmortem submersion. In this study, the identification and succession of epinecrotic bacterial communities on vertebrate remains were described during decomposition in a temperate headwater stream during two seasons (summer and winter). Bacterial communities were characterized with 454 pyrosequencing and analyzed at phyletic and generic taxonomic resolutions. There was a significant increase in genera richness over decomposition during both seasons. Additionally, multivariate statistical modeling revealed significant differences in bacterial communities between seasons at both taxonomic resolutions and siginificant genera differences among sampling days within each season, suggesting a succession of these communities. These data are the first to describe aquatic bacterial succession using high-throughput metagenomic sequencing on vertebrate remains submerged in a freshwater habitat, and provide initial evidence for their potential use in forensic investigations.     

Forensic analysis of soils using single arbitrarily primed amplification and high throughput sequencing

Soil is a remarkably complex, diverse, ubiquitous, and easily transferred material which can reveal highly useful information to assist forensic investigations. In spite of its potential usefulness, the use of genetic soil analysis appears to be currently underestimated in forensic practice. Herein we report on the use of single arbitrarily primed amplification followed by high throughput sequencing of DNA fragments for the comparison of soil samples. The composition and functional attributes of soil microbial communities from three different locations were compared and shown to be different based on the metagenomic sequencing data obtained.     

An Analysis of Systematic Elemental Changes in Decomposing Bone

The aim of this pilot study was to investigate compositional changes in bone during decomposition. Elemental concentrations of barium, calcium, iron, potassium, magnesium, zinc and phosphorus in porcine bone (as an experimental analog for human bone) were analyzed by inductively coupled plasma optical emission spectroscopy (ICP‐OES). The samples were taken from porcine bone subjected to shallow burial and surface depositions at 28‐day intervals for a period of 140 days. Results indicated that ICP‐OES elemental profiling has potential to be developed as a forensic test for determining whether a bone sample originates from the early stages of soft tissue putrefaction. Significant changes in iron, sodium and potassium concentrations were found over 140 days. These elements are known to be primarily associated with proteins and/or tissue fluids within the bone. Changes in their respective concentrations may therefore be linked to dehydration over time and in turn may be indicative of time since deposition.     

RNA cell typing and DNA profiling of mixed samples: Can cell types and donors be associated?

Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling.     

The use of organic and inorganic impurities found in MDMA police seizures in a drug intelligence perspective

Traditional forensic drug profiling involves numerous analytical techniques, and the whole process is typically costly and may be time consuming. The aim of this study was to investigate the possibility of prioritising techniques utilised at the Australian Federal Police (AFP) for the chemical profiling of 3,4-methylenedioxymethylamphetamine (MDMA). The outcome would provide the AFP with the ability to obtain more timely and valuable results that could be used in an intelligence perspective. Correlation coefficients were used to obtain a similarity degree between a population of linked samples (within seizures) and a population of unlinked samples (between different seizures) and discrimination between the two populations was ultimately achieved. The results showed that gas chromatography–mass spectrometry (GC–MS) was well suited as a single technique to detect links between seizures and could be used in priority for operational intelligence purposes. Furthermore, the method was applied to seizures known or suspected (through their case information) to be linked to each other to assess the chemical similarity between samples. It was found that half of the seizures previously linked by the case number were also linked by the chemical profile. This procedure was also able to highlight links between cases that were previously unsuspected and retrospectively confirmed by circumstantial information. The findings are finally discussed in the broader forensic intelligence context, with a focus on how they could be successfully incorporated into investigations and in an intelligence-led policing perspective in order to understand trafficking markets.     

Novel cellular signatures for determining time since deposition for trace DNA evidence

With the increase in sensitivity of DNA profiling, questions about how and when the DNA was deposited have become a driving issue in forensic cases. To address this, we propose a novel method to determine time since deposition of trace DNA samples based on morphological and autofluorescence properties of individual epithelial cells which can change as the sample ages. To develop this signature, a series of trace DNA samples were generated by contact/handling a substrate and then allowed to age anywhere between one day and more than one year prior to collection. Imaging flow cytometry (IFC) was then used to characterize the morphology and autofluorescence profiles of individual cells within each sample followed by multivariate modelling and predictive classification.Resultsshowed that epithelial cell populations could be classified with high accuracy (∼90%) into one of three time-since-deposition groups: < 1 week, between 1 week and 2 months, and > 2months. Differences across age groups were largely driven by decreases in brightfield contrast and increases in the intensity of autofluorescence. To further test this approach for forensic casework, 47 individual donor cell populations spanning each time deposition group were classified blindly against the remaining data set. Samples containing at least 75 cells and a posterior probability greater than 0.90 showed classification accuracies ∼95%. Accuracies for individual time groups were 97% (<1 week), 92% (1week-2months), 98% (>2 months) with an average posterior probability for all time groups ∼0.96. This indicates that autofluorescence and morphological analyses may provide probative information regarding time since deposition for many types of trace DNA samples in forensic casework.     

High‐throughput Sequencing of Trace Quantities of Soil Provides Reproducible and Discriminative Fungal DNA Profiles

High‐throughput sequencing (HTS) offers improved resolution between forensic soil samples by characterizing individual taxa present; however, the heterogeneous distribution of taxa in soils, and limited quantity of material available, may hinder the reliability of HTS in casework. Using HTS of the internal transcribed spacer, we examined the effect of soil mass (50, 150, and 250 mg) on fungal DNA profiles, focusing on reproducibility and discriminatory power between close proximity soils, and samples with similar textural classification. The results show that reduced soil mass had no significant effect on sample differentiation and that 150 mg soil provides the most reproducible DNA profiles across different soil types. In addition, Ascomycota was identified as a robust fungal target for forensic intelligence as this phylum was detected consistently across all samples regardless of sample quantity. Overall, this study highlights the value of trace quantities of soil for use in forensic casework.