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.     

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.     

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.     

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 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.     

Forensic comparison of soils by bacterial community DNA profiling

This preliminary investigation has shown that a soil microbial community DNA profile can be obtained from the small sample of soil recovered from the sole of a shoe, and from soil stains on clothing. We have also shown that these profiles are representative of the site of collection and therefore could potentially be used as associative evidence to prove a link between suspects and crime scenes. Soil community profiles were obtained using the T-RFLP fingerprinting method that uses fluorescent primer technology and semi-automated analysis techniques similar to those used in human DNA profiling in forensic laboratories.     

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.     

Spatial and temporal influences on bacterial profiling of forensic soil samples

Bacterial content may be helpful in differentiating forensic soil samples; however, the effectiveness of bacterial profiling depends on several factors, including uniqueness among different habitat types, the level of heterogeneity within a habitat, and changes in bacterial communities over time. To examine these, soils from five diverse habitats were tested over a 1 year period using terminal restriction fragment length polymorphism (TRFLP) analysis. Soil samples were collected at central locations monthly, and 10 feet in cardinal directions quarterly. Similarity indices were found to be least related among habitats, while the greatest bacterial similarities existed among collection locations within a habitat. Temporally, however, bacterial content varied considerably, and there was substantial overlap in similarity indices among habitats during different parts of the year. Taken together, the results indicate that while bacterial DNA profiling may be useful for forensic soil analysis, certain variables, particularly time, must be considered.     

微生物物证检验

面对21世纪生物犯罪或生物恐怖活动的新挑战,物证鉴定的新专业--微生物物证检验将成为执法部门侦查和起诉生物犯罪必不可少的手段。微生物物证检验以用作犯罪武器的各种微生物为检验对象,获得微生物种类和能够提供来源信息的菌毒株细致分型结果,达到提供犯罪侦查线索和法庭证据的目的。本文综述了微生物物证检验的定义、特征、技术应用以及美国近年来在微生物物证检验的实践和值得借鉴的成功经验。并建议我国物证鉴定实验室应积极开展研究,建立能够满足生物犯罪侦查需求的微生物物证检验能力。     

Bioinformatics Approach to Assess the Biogeographical Patterns of Soil Communities: The Utility for Soil Provenance,

Soil DNA profiling has potential as a forensic tool to establish a link between soil collected at a crime scene and soil recovered from a suspect. However, a quantitative measure is needed to investigate the spatial/temporal variability across multiple scales prior to their application in forensic science. In this study, soil DNA profiles across Miami‐Dade, FL, were generated using length heterogeneity PCR to target four taxa. The objectives of this study were to (i) assess the biogeographical patterns of soils to determine whether soil biota is spatially correlated with geographic location and (ii) evaluate five machine learning algorithms for their predictive ability to recognize biotic patterns which could accurately classify soils at different spatial scales regardless of seasonal collection. Results demonstrate that soil communities have unique patterns and are spatially autocorrelated. Bioinformatic algorithms could accurately classify soils across all scales with Random Forest significantly outperforming all other algorithms regardless of spatial level.     

Soil forensics: How far can soil clay analysis distinguish between soil vestiges?

Soil traces are useful as forensic evidences because they frequently adhere to individuals and objects associated with crimes and can place or discard a suspect at/from a crime scene. Soil is a mixture of organic and inorganic components and among them soil clay contains signatures that make it reliable as forensic evidence. In this study, we hypothesized that soils can be forensically distinguished through the analysis of their clay fraction alone, and that samples of the same soil type can be consistently distinguished according to the distance they were collected from each other. To test these hypotheses 16 Oxisol samples were collected at distances of between 2 m and 1.000 m, and 16 Inceptisol samples were collected at distances of between 2 m and 300 m from each other. Clay fractions were extracted from soil samples and analyzed for hyperspectral color reflectance (HSI), X-ray diffraction crystallographic (XRD), and for contents of iron oxides, kaolinite and gibbsite. The dataset was submitted to multivariate analysis and results were from 65% to 100% effective to distinguish between samples from the two soil types. Both soil types could be consistently distinguished for forensic purposes according to the distance that samples were collected from each other: 1000 m for Oxisol and 10 m for Inceptisol. Clay color and XRD analysis were the most effective techniques to distinguish clay samples, and Inceptisol samples were more easily distinguished than Oxisol samples. Soil forensics seems a promising field for soil scientists as soil clay can be useful as forensic evidence by using routine analytical techniques from soil science.     

Soils of Contrasting pH Affect the Decomposition of Buried Mammalian (Ovis aries) Skeletal Muscle Tissue

Abstract:  Little is known about the effect of edaphic conditions on the decomposition of buried mammalian tissues. To address this, we set up a replicated incubation study with three fresh soils of contrasting pH: a Podsol (acidic), a Cambisol (neutral), and a Rendzina (alkaline), in which skeletal muscle tissue (SMT) of known mass was allowed to decompose. Our results clearly demonstrated that soil type had a considerable effect on the decomposition of SMT buried in soil. Differences in the rate of decomposition were up to three times greater in the Podsol compared with the Rendzina. The rate of microbial respiration was correlated to the rate of soft tissue loss, which suggests that the decomposition of SMT is dependent on the microbial community present in the soil. Decompositional by-products caused the pH of the immediate soil environment to change, becoming more alkaline at first, before acidifying. Our results demonstrate the need for greater consideration of soil type in future taphonomic studies.     

Predictive Soil Provenancing (PSP): An Innovative Forensic Soil Provenance Analysis Tool

Soil is a common evidence type used in forensic and intelligence operations. Where soil composition databases are lacking or inadequate, we propose to use publicly available soil attribute rasters to reduce forensic search areas. Soil attribute rasters, which have recently become widely available at high spatial resolutions, typically three arc‐seconds (~90 m), are predictive models of the distribution of soil properties (with confidence limits) derived from data mining the inter‐relationships between these properties and several environmental covariates. Each soil attribute raster is searched for pixels that satisfy the compositional conditions of the evidentiary soil sample (target value ± confidence limits). We show through an example that the search area for an evidentiary soil sample can be reduced to <10% of the original investigation area. This Predictive Soil Provenancing (PSP) approach is a transparent, reproducible, and objective method of efficiently and effectively reducing the likely provenance area of forensic soil samples.     

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.     

The role of forensic geoscience in wildlife crime detection

The increase in both automation and precision in the analysis of geological materials has had significant impact upon forensic investigations in the last 10 years. There is however, a fundamental philosophical difference between forensic and geological enquiry. This paper presents the results of forensic geoscientific investigations of three cases of wildlife crime. Two cases involve the analysis of soils recovered after incidents of illegal badger baiting in the United Kingdom. The third case involves the illegal importation of Eleonora's Falcon (Falco eleonorae) into the United Kingdom from the Mediterranean. All three cases utilise the analysis of soils by a variety of physical, chemical and biological techniques. These involve mineral and grain size analyses, cation and anion compositions, pH, organic content and pollen analysis. The independent analysis undertaken by specialists in each of these three main fields conclude firstly, that there is a significant similarity between sediments taken at the crime site at both badger setts and with sediments recovered from various spades, shovels and clothing belonging to suspects and secondly, that the soils analysed associated with the removal of the falcon eggs in the Mediterranean contained characteristics similar in many respects to the soils of the breeding areas of F. eleonorae on the cliffs of Mallorca. The use of these independent techniques in wildlife crime detection has great potential given the ubiquitous nature of soils and sediments found in association with wildlife sites.     

Developing and Testing a Soil Property Database for Forensic Applications in Southern California

The research sought to develop and test a forensic database of surface soil variability within previously mapped geologic and soil units in southern California. This type of database could be used to link suspects to crime scenes or determine source locations of soil sample evidence. Variability was evaluated using (i) color, (ii) magnetic susceptibility, and (iii) particle‐size distribution. Soil properties were analyzed for their ability to discriminate source areas using stepwise discriminant analysis. The percent correct predictions for geologic unit groups ranged from 30% to 100%. A blind study experiment matched four of the 18 samples to their unit of origin with the first choice by stepwise discriminant analysis, and eight were matched as second and third choices. The probability of selecting the appropriate unit of origin increased by 54% over random chance and eliminated as much as 99% of the field area as a potential search location.     

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.     

Understanding Variations of Soil Mapping Units and Associated Data for Forensic Science

Soil samples have potential to be useful in forensic investigations, but their utility may be limited due to the inherent variability of soil properties, the wide array of analytical methods, and complexity of data analysis. This study examined the differentiation of similar soils based on both gross (texture, color, mineralogy) and explicit soil properties (elemental composition, cation exchange, Fe‐oxyhydroxides). Soils were collected from Fallbrook and adjacent map units from Riverside and San Diego Counties in California. Samples were characterized using multiple techniques, including chemical extracts, X‐ray diffraction (XRD), and Fourier transform infrared spectroscopy. Results were analyzed using multiple analytical approaches to compare counties and land uses. Some analyses (XRD, extractions) were better at distinguishing among samples than others (color, texture). Ratios of rare earth elements were particularly useful for distinguishing samples between counties. This potential to “fingerprint” soils illustrates the usefulness of a comprehensive soil database for criminal investigators.     

Limitations and recommendations for successful DNA extraction from forensic soil samples: A review

Soil is commonly used in forensic casework to provide discriminatory power to link a suspect to a crime scene. Standard analyses examine the intrinsic properties of soils, including mineralogy, geophysics, texture and colour; however, soils can also support a vast amount of organisms, which can be examined using DNA fingerprinting techniques. Many previous genetic analyses have relied on patterns of fragment length variation produced by amplification of unidentified taxa in the soil extract. In contrast, the development of advanced DNA sequencing technologies now provides the ability to generate a detailed picture of soil microbial communities and the taxa present, allowing for improved discrimination between samples. However, DNA must be efficiently extracted from the complex soil matrix to achieve accurate and reproducible DNA sequencing results, and extraction efficacy is highly dependent on the soil type and method used. As a result, a consideration of soil properties is important when estimating the likelihood of successful DNA extraction. This would include a basic understanding of soil components, their interactions with DNA molecules and the factors that affect such interactions. This review highlights some important considerations required prior to DNA extraction and discusses the use of common chemical reagents in soil DNA extraction protocols to achieve maximum efficacy. Together, the information presented here is designed to facilitate informed decisions about the most appropriate sampling and extraction methodology, relevant both to the soil type and the details of a specific forensic case, to ensure sufficient DNA yield and enable successful analysis.