Multi-technique comparison of source and primary transfer soil samples: an experimental investigation.

The comparison of soil samples is of long-standing and increasing interest in forensic science. Routine comparisons should involve the use of several techniques in combination. A wide range of analytical techniques can be used, choice being dependent on several factors including sample size and character, time constraints and cost limitations. In this paper results are presented for experiments carried out to test the effectiveness of four of the available analytical techniques (spectro-photometric colour determination, laser diffraction particle size analysis, stable isotope analysis and chemical element analysis) used to compare single source and primary transfer soil samples. Four soil types and five footwear types were used. All four techniques showed excellent precision and good resolving power between soil types. Only relatively small differences were obtained between source and transferred soil samples in terms of colour, stable carbon and nitrogen isotope ratios and elemental chemistry. Slight but significant differences were found in grain size, indicating that the primary transfer process is to some extent grain size selective.     

Sediment fingerprints: a forensic technique using quartz sand grains.

The analyses of 738 soil and sediment samples taken during the investigation of 20 forensic cases are combined in a database for retrospective comparisons. Quartz sand grain surface textures are combined into grain types which are analysed at one of five orders of classification. This classification also provides frequency data on the number of grain types found in a sample, the individuality of the quartz grain type assemblages and quartz grain type ratios. The technique, developed primarily as an exclusion mechanism, suggests possibilities, in some circumstances, that significant interpretations can be achieved at different orders of classification. The technique provides excellent exclusionary results and, when combined with other independent lines of analysis, is a powerful tool in forensic interpretation because it uses quartz as its discriminatory mineral. The technique also highlights the potential for computer image recognition analysis.     

Particle size analysis of sediments, soils and related particulate materials for forensic purposes using laser granulometry

Particle size is a fundamental property of any sediment, soil or dust deposit which can provide important clues to nature and provenance. For forensic work, the particle size distribution of sometimes very small samples requires precise determination using a rapid and reliable method with a high resolution. The Coulter trade mark LS230 laser granulometer offers rapid and accurate sizing of particles in the range 0.04-2000 microm for a variety of sample types, including soils, unconsolidated sediments, dusts, powders and other particulate materials. Reliable results are possible for sample weights of just 50 mg. Discrimination between samples is performed on the basis of the shape of the particle size curves and statistical measures of the size distributions. In routine forensic work laser granulometry data can rarely be used in isolation and should be considered in combination with results from other techniques to reach an overall conclusion.     

The Use of a Sequential Extraction Technique to Characterize Soil Trace Evidence Recovered from a Spade in a Murder Case in Brazil*

Soil trace evidence can be useful in criminal investigations. A homicide which had occurred in South Brazil been concluded through the courts with a guilty conviction. A spade with soil traces adhering to it was seized from the confessed killer's house, it having been established that it had been used to bury parts of the victim's body. In the context of this confession, it provided an opportunity to test a protocol of analysis and verify the potential of discriminate soil sample analysis in such case works. This allowed us to test the practice of sequential analysis which had been developed for forensic case works in Brazil, with three sequential extractions: (i) 0.2 mol/L pH 3.0 ammonium oxalate; (ii) dithionite–citrate–bicarbonate; and (iii) 0.5 mol/L NaOH. It was possible to predict the sequence of events related to the homicide by using the sequential extraction technique and to conclude that: (i) the A horizon soil from the burial location of the torso was found to be very similar to the soil samples which had been recovered from the spade, which was able to be established despite there only being a small amount of soil adhering to the spade; (ii) the location where the legs were buried contributed a low amount of soil adhering to the spade. Therefore, it is suggested that, where possible, sequential extractions should be prioritized from a questioned sample to best provide information about the likely sequence of contact places and this test likely scenarios and criminal events.     

Application of mtDNA SNP analysis in forensic casework

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.     

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.     

Screening of soil evidence by a combination of simple techniques: validity of particle size distribution.

The validity of the determination of particle size distribution for forensic soil identification was studied using 73 soil samples collected from a 20 km x 15 km area. Soil particles were first divided into two fractions by wet sieving with a sieve with a 0.05 mm aperture size. Dry sieving was used to analyze the coarse particle fraction and a particle size analyzer was used for the fine particle fraction. The number of particle size classes measured by sieving could be reduced to three, <0.05, 0.05-0.2, and 0.2-2 mm, which enabled 87.9% of the soil samples to be discriminated. Measurements made with the particle size analyzer on the fine particle fraction increased the discriminating power to 95.9%. The use of particle size analysis by sieving in combination with color examination allowed us to discriminate 99.5% of the soil samples, even when particle size analysis of the fine particle fraction was not carried out.     

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.     

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.     

Particle size analysis of soils under simulated scene of crime conditions: the interest of multivariate analyses

Two simulated scenes of crime have been studied. Soil traces adhering to boots, sport shoes and tissues have been compared with control samples using particle size analysis. Comparisons of percentage of particles per class interval and multivariate analyses were used to determine how the size distribution of each suspect sample varied compared to the original distribution. A loss of coarse fractions characterized most of the suspect samples even though they were derived from soils having different distributional patterns. However, the differences between control and suspect samples were found to be more important for soils dominated by extreme size classes than for soils rich in medium size particles. The differences among soil samples from the same source were found to be restricted to the extreme size classes. The finding of significant differences within size classes ranging from 1 to 0.063 mm should be then considered as a dissimilarity sign between trace sample and hypothetic control soil.     

Particle size analysis of soils under simulated scene of crime conditions: the interest of multivariate analyses

Two simulated scenes of crime have been studied. Soil traces adhering to boots, sport shoes and tissues have been compared with control samples using particle size analysis. Comparisons of percentage of particles per class interval and multivariate analyses were used to determine how the size distribution of each suspect sample varied compared to the original distribution. A loss of coarse fractions characterized most of the suspect samples even though they were derived from soils having different distributional patterns. However, the differences between control and suspect samples were found to be more important for soils dominated by extreme size classes than for soils rich in medium size particles. The differences among soil samples from the same source were found to be restricted to the extreme size classes. The finding of significant differences within size classes ranging from 1 to 0.063 mm should be then considered as a dissimilarity sign between trace sample and hypothetic control soil.     

载体法检验微量检材DNA

目的解决微量生物检材DNA检验难题。方法采用载体法对已知微量血样DNA进行检验，再与目前高灵敏度的Chelex-100提取的DNA检验的相应结果进行比较。结果载体法针对微量血样的DNA检验。不但能得到正确的STR分型结果．同时在检验灵敏度上比Chelex-100法高出2倍以上。结论载体法可提高微量生物栓材中DNA检出率，且操作简单，在法医检案工作中具有较高应用价值。     

Comparative Forensic Soil Analysis of New Jersey State Parks Using a Combination of Simple Techniques with Multivariate Statistics,

This study has shown that the combination of simple techniques with the use of multivariate statistics offers the potential for the comparative analysis of soil samples. Five samples were obtained from each of twelve state parks across New Jersey in both the summer and fall seasons. Each sample was examined using particle‐size distribution, pH analysis in both water and 1 M CaCl₂, and a loss on ignition technique. Data from each of the techniques were combined, and principal component analysis (PCA) and canonical discriminant analysis (CDA) were used for multivariate data transformation. Samples from different locations could be visually differentiated from one another using these multivariate plots. Hold‐one‐out cross‐validation analysis showed error rates as low as 3.33%. Ten blind study samples were analyzed resulting in no misclassifications using Mahalanobis distance calculations and visual examinations of multivariate plots. Seasonal variation was minimal between corresponding samples, suggesting potential success in forensic applications.     

STR analysis of artificially degraded DNA-results of a collaborative European exercise

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.     

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.     

Evaluation of microbial soil identity in forensic science

Microbiologic analysis of different soil samples was undertaken to assess the value of this method for forensic purposes. Three groups of micro-organisms were isolated on selective media: bacteria, fungi, and actinomycetes. Comparison of the morphology of the colonies led to the conclusion of non identity of the soil samples under examination. The method was applied to soil traces on articles of clothing, trousers, shoes, and stockings. It was found that the soil did not originate from the suspected scene of a crime. Examination of the microbiologic soil composition does not provide an absolute certainty of identity, but it is highly reliable.     

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.     

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.     

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.     

A Test of Sex Estimation in Subadults Using the Elevation of the Auricular Surface from Four Samples of Known Age and Sex

Biological sex is foundational to the work of forensic anthropologists and bioarcheologists. The lack of reliable biological sex estimation methods for subadults has, thus, greatly limited forensic and bioarcheological analyses. Auricular surface elevation showed promise as a subadult sex estimation method in previous studies. This study examined two auricular surface elevation evaluation methods on four subadult samples of known age, sex, and ancestry. Samples were scored as “male,” “female,” or “indeterminate” and results were examined with chi‐square analysis. No consistent sex estimation pattern, accuracy, or predictive value was produced between samples. Only one test was significant using Fisher's exact test analysis (FET = 7.501, p < 0.022): the composite approach on the Hamann‐Todd sample. While age, sample size, or developmental factors may play a role in these results, clearly sample variation does as well. This study found auricular surface elevation was not a useful subadult sex estimation method.