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FP7

MISAFE Résumé de rapport

Project ID: 313149
Financé au titre de: FP7-SECURITY
Pays: Israel

Final Report Summary - MISAFE (The Development and Validation of Microbial Soil Community Analyses for Forensics Purposes)

Executive Summary:
The ubiquity, complexity, heterogeneity and transferability of soil make it make it valuable in forensic investigations, to yield clues as to the origin of an unknown sample, or to compare samples from a suspect or object with control samples from a scene. Soil characteristics are affected by soil origin, history, environment and management: these characteristics are defined by variables that are measurable. This forms the basis upon which measurements of soil variables are indicative of soil provenance, hence the interest of such measurements for forensics science. To date, soil is an underused resource. This is mainly due to the need for considerable expertise required by some of the methods used to analyse soil samples, and for the interpretation of the resulting data thereof; for example, mineralogy and palynology.
Another soil resource that is almost unexploited in forensics to date is the microbial life forms found in soil. The microbial content of soils is enormous, above 109 cells per gram; its diversity is still uncharacterized, because of its sheer size, which has been estimated to span from 10 000 and 10 million bacterial species per gram. This great diversity depends upon soil type, soil cover, and environmental conditions; hence it potentially contains a wealth of information pertinent to the forensics investigator. However, access to this great diversity using classical microbiological tools is largely limited by biotic and abiotic constrains: most soil bacteria (more than 99%) do not grow in the laboratory on microbiological media. The major technological developments that have occurred in the last two decades in the field of DNA sequencing and in bioinformatics have completely revolutionized how environmental microbiology/microbial ecology research is performed, and our understanding of the microbial realm. Extensive data sets of the diversity of soil bacteria can be obtained by analyzing phylogenetic marker genes such as the 16S rRNA gene. This information requires that DNA is extracted and purified from the soil, amplified using PCR, and the resulting amplicon analysed to reveal the sequence diversity it contains. This is achieved by examining a banding pattern in a gel-based assay or by directly sequencing individual molecules in the amplicon. The resulting data have in any case to the treated, and analysed, and if different samples are compared, submitted to appropriate statistical analyses.
The challenges of applying information on soil microbial diversity in forensic investigation are numerous: so far, no solid theory on the biogeography of microbes exists, and whether rules such as distance-decay relationship or universal distribution of species complemented by local selection apply are controversial; soil diversity is huge and as such, no one method to obtain DNA in quantity and quality sufficient for analysis has been developed; many other gene markers exist, which may be of interest to replace or complement the 16S rRNA gene approach but they have not yet been tested; PCR introduces biases into the amplicon, and not one protocol fits the quantity/quality of DNA samples obtained from soils; many technologies exist to analyse the amplicon and their relative performance in analysis soils samples is not known; the ability to discern between soil types and within sites in the same soil type, and the influence of seasons of soil microbial community structure and their applicability to the forensics context has barely been investigated, and; the legal context of the use of microbial analysis for soil forensics has not been determined. These challenges form the basis of the objectives of MiSAFE.
Accordingly, MiSAFE’s objectives were to provide the forensic practitioner with a microbial genetic profiling tool that will bring soil into routine forensic investigation and judicial proceedings.
To achieve these objectives, MiSAFE was built upon eight work packages, each dedicated to a particular large task but all tightly interconnected, providing easy and efficient flow of information between the disciplines and between the partners. Management (WP7) was the responsibility of the HUJI coordinator but this was largely shared by ECL who performed most of the financial aspects. Also, two external forensic experts were appointed to the project advisory group and provided essential professional inputs.
The extraction of DNA from soil was addressed by WP1. A panel of extraction methods was evaluated on a range of soils using different physico-chemical and biological criteria for assessing quantity and quality of the extracted DNA. This resulted in recommendations for a “best performing kit” under the tested conditions.
WP2 built a series of guidlines for assessing a scene, sampling, transporting and storing soil DNA. This series of guidelines were based on surveys of forensics laboratories around the world as well as on experience, experimental and field work. WP2 also constructed a mock-crime scenario and coordinated the analysis of the relevant soil samples by 12 laboratories, including MiSAFE and non-MiSAFE partners, using biological and non-biological methods. Biological (DNA-based) alone or in conjunction with other approaches proved to be able to reliably classify the soil samples.
WP3 assessed the validity of a set of markers and compared them to the 16S rRNA gene “gold standard”. None was as powerful as the 16S rRNA gene approach. WP3 also evaluated the ability of Ribosomal Intergenic Space Analysis (RISA), of phylogenetic 16S rRNA- based microarray and Miseq sequencing (Illumina) as well as of QPCR and enzymatic digestion to discern between soil types. While RISA and Miseq performed well, microarrays did not, suggesting that this technology does not fulfill forensic requirements.
WP4 explored the ability to discern between and within soil types using TRFLP and other DNA-based technologies. It showed that TRFLP could be greatly optimized, increasing its spatial resolution between sites. A main finding was that most technologies (RISA, TRFLP, high throughput sequencing and analysis of organics) all perform well in discriminating between soil types. WP4 also showed that the structure of soil bacterial communities is greatly affected by season in Israel where the weather fluctuates sharply during the year. Seasonal effects appear to be greater than soil type effects. Manipulation of soil samples to mimic and mitigate such effects was not successful but suggested that the approach is sound but needs further research for validation.
WP5 led the development of a fully-fledged bioinformatics workbench for analysis of environmental bacterial DNA samples. The software was developed, tested and launched commercially in the USA. Further, WP5 improved upon existing geoforensics databases and created guidance for DNA-based geoforensic platforms.
In WP6, the legal aspects of soil bacterial forensics were investigated under the various types of law prevailing in Europe, producing a thorough comparison of their frameworks and implication for forensic application of a soil DNA approach.. It was concluded that DNA-based soil forensics do not constitute a major change upon the existing technologies and that its application at the EU should not create major difficulties within civil law countries while further validation is required before it is used as evidence in common law countries. WP6 produced a set of protocols and recommendations using ISO criteria.
Finally, dissemination of the outputs of the MiSAFE research was carried out under WP8. A website was established and there were over 60 related events (talks, publications, interviews for example). The international impact was considerable with successful collaboration with European and global networks.

Project Context and Objectives:
Soil is ubiquitous and heterogeneous, with soil characteristics being affected by soil origin, history, environment and management. These characteristics make soil a useful material for forensic applications. However, this is currently not the case throughout the whole of Europe, or the world. The aim of MiSAFE is to develop a practical approach by which the microbial aspects of soil can be considered for use in forensic investigations within a wider European context than is currently the situation.
Soils in forensic investigations
Soil complexity, heterogeneity and transferability make it valuable in forensic investigations, to yield clues as to the origin of an unknown sample, or to compare samples from a suspect or object with a crime scene (1). In a few countries soil analysis is used in legal submissions on issues from site verification, to body decomposition (2). High profile cases in the UK have used soil as evidence, and made use of data on physical and elemental data on soils within Geographic Information Systems (GIS) for use in intelligence, drawing on national data held by the James Hutton Institute (3) from the National Soils Archive and Database of Scotland. Yet, to date, the application or use of soil information in criminal investigations has been limited. This is often due to a lack of awareness of the important role such information can play, and constraints of the current methodology of soil analysis, which relies upon details of its chemical and mineralogical composition – and on expert knowledge in interpretation in particular, examining features such as soil type and colour as well as its elemental, mineralogical, and organic profile characteristics (4), demanding considerable expertise. Furthermore, the resolution which can be obtained from use of these tools is sometimes limited by inability to distinguish between locations of samples. A few cases have made use of microbial analyses, such as in Italy, where genetic fingerprinting of the bacterial community, coupled with elemental analysis provided key evidence for concluding the case in the judicial court (5) in a murder case. Similarly, in Holland, soil microbial characteristic information is integrated with elemental and palynological data in many cases of serious crimes investigation. In addition, in Spain, aspects of microbial characteristics are routinely used in case work. In the UK, occasionally expert microbiological expertise is used in case work, e.g. fungal characterisation comparing a questioned sample with a grave site location.
Soil as a rich medium rich in microbial information
The microbial content of soils is enormous, above 109 cells per gram; its diversity is still uncharacterized, because of its sheer size, which has been estimated to span from 10 000 and 10 million bacterial species per gram (6, 7). However, access to this great diversity is largely limited by biotic and abiotic constrains: most soil bacteria (more than 99%) do not grow in the laboratory on microbiological media. However, with powerful molecular technologies soil bacterial DNA can be isolated and characterized, thereby overcoming the cultivation barrier. Extensive data sets of the diversity of soil bacteria can be obtained using phylogenetic marker genes such as the 16S rRNA gene. Nevertheless, localization of some populations in protected and barely accessible microsites inside the soil matrix, irreversible adsorption of part of bacterial cells and DNA onto soil particles such as clay, and interference of DNA with co-extracted humic acids contribute to limiting DNA extraction yields (8); because of this, and other technical issues such as PCR biases and sequence read length, and comprehensive coverage of soil, any soil-bacterial diversity is still to be achieved. While some populations in the community may have a high abundance, most of the diversity is composed of populations of very low abundance, the so called ‘rare biosphere’.
Thus, the community structure of a bacterial community could be a powerful tracer of the origin of a soil sample. Although the potential for the forensic use of soil microbial communities is apparent, its practical use has been very limited, a main reason for that being a lack of clear guidelines as to the appropriate technologies, the resolution, reliability and robustness of the results in addition to lack of a comprehensive database of the values and associated knowledge for case interpretation.

Project Results:
The MiSAFE has produced results relevant to science, technology and society. The main points for each of these are:
Science
Forensics: The demonstration that soils can be differentiated or compared using their microbial communities as indicators
Microbial ecology: reinforcing theory on microbial distribution; setting context of microbial community structure in relation to environmental parameters, including soil chemistry, geography and climate; Providing a measures of housekeeping genes (phylogenetically universal genes in Bacteria) diversity; finding that simple soil storage preserves DNA for long periods of time
Statistics: adapting and developing novel statistical approaches for soil sample comparison.

Technology
Optimized soil DNA extraction procedures
In depth comparison of microbial community structure analytical methods and their adaptation to specific issues
A software package for the analysis of microbiomes from any environment based on high-throughput sequencing or fingerprinting approaches
An integrated approach to include DNA data in geographic databases

WP results (Tables and figures and in the pdf)
Table A provides a summary of the WP's achievements.

WP01: Sample processing and protocol development

Involved partners 01 HUJI, 02 LIB, 04 ECL, 05 JHI
Objectives
To define a standardized procedure for the assessment of the quantitative and qualitative DNA features
To evaluate a wide spectrum of available commercial kits and reagents or traditional methods for the extraction and purification of nucleic acids from environmental samples
To select the best performing protocol for an evaluation in forensic scenarios

WP1 aims to draw up a comprehensive evaluation of DNA extraction methods of total microbial DNA, including bacteria and fungi, from environmental soil samples.

Main results and achievements
A panel of DNA extraction methods was compared (Table 1) using various descriptors for which a normalization procedure was developed (Table 2). These kits and descriptors were applied onto a panel of various soils from the UK representing a variety of soil types (Table 3).
The descriptors included standard DNA evaluation parameters for quantity and purity as measured by spectrophotometric measurements of OD260/OD280 and OD260/OD230 ratios and novel procedures , including the definition of a standard DNA extract to be used as an objective evaluation of DNA quantitation methods (internal verification of the methodology); the application of fluorometric measurement, and; a method to evaluate the degree of sample contamination through the efficiency of PCR amplification.
It was concluded that quantitative and qualitative features of a soil DNA extract can be well described by documenting the following technical check-list:
DNA extraction meta-data (soil sample information, dry matter and water content of the soil, reagents and extraction protocol, DNA storage conditions, …)
DNA solvent (ex Tris HCl buffer, ultra-pure water, TE10, 1 …)
DNA concentration (ng/µl) estimated with fluorimetric quantitation method
OD260/OD280 ratios
OD260/OD230 ratios
DNA size and integrity using an electrophoretic separation method
DNA purity estimated by homologous and/or heterologous PCR assay
The descriptors were normalized to allow a synthetic and transversal graphical view of the results of DNA extraction on a given soil using the 10 extraction methods (Table 3). A scoring with differential weighting of the various quantitative parameters was then included along with the normalization to allow direct comparison of the results of a given extraction method on the different soils (Table 4).

The scoring was applied to nine extraction methods and the four UK soils (Table 5). This method is flexible, enabling adding parameters like time (min), an appreciation of operator time required to produce a single DNA extraction; delay (min) which integrates incubation times, causing delay in the recovery of DNA extracts without consuming operator time, or; cost (€), the estimated cost for a single DNA extraction can be included. A graphical representation was developed and applied. Based on the evaluation of the four tested soils, the NucleoSpin kit could be selected as a reference method since it provided fully usable for PCR-based experiments, with an acceptable medium cost and a good delay of DNA recovery (Fig.1).

The three most efficient kits, namely the NucleoSpin, the Norgen and the PowerLyser kits, were then selected for a second round of evaluation on an extended panel of soils from Israel that represent very different soil types and climatic conditions.
The three most efficient kits, namely the NucleoSpin, the Norgen and the PowerLyser kits, were then selected for a second round of evaluation on a panel of soils from Israel that represent very different soil types and climatic conditions (renzina, terra rossa, desert skeletal soil and sand). This round of evaluation revealed that the NucleoSpin and Norgen kits, which gave the most satisfactory results on the first soil panel, did not allow a satisfactory recovery of DNA extracts from the soils from Israel. In contrast, the PowerLyser PowerSoil kit yielded high quality DNA extract for the four soils from Israel.
Considering the whole panel of eight different soils (Fig.2 ) the PowerLyser PowerSoil kit gave the best overall results, except for the peaty gley Ardallie soil.

Conclusions: Based on the the overall results on an extended panel of eight highly contrasted soils, the PowerLyser PowerSoil kit can be recommended as a reference method to produce high-quality DNA for PCR-based experiment. A reduction of the amount of treated soil material (about 100 mg instead on the manufacturer recommended 250 mg) should be adopted, minimizing the risk of producing low quality DNA on some recalcitrant soil samples.

WP02: Sample collection and storage

Involved partners 01 HUJI, 02 LIB, 04 ECL, 05 JHI, 06 MOPS/INP, 07 GC
Objectives:
To review and define key technical, experimental and statistical parameters necessary to develop a suitable sampling strategy
To develop a strategy for sampling crime scene and comparator samples in relation to replication
To develop a strategy for sampling items
To test the impact of condition, sample size, and depth on microbial analysis methods
To test the impact of sample storage on quality of sample analyses
Assess the approach developed for the Rothamsted soil on a range of contrasting soil types
Develop a set of Best Practice and Standard Operating Procedures for the sampling, collection, storage and analysis of trace soil biological evidence
Develop guidelines for the best use of soil intelligence and evidence by investigators across the range of investigative situations.

Main results and achievements
Work in WP2 has produced:
A survey of soil sampling and forensics use in Europe, providing an assessment of current procedures and interest in soil forensics in Europe.
The design of a sampling template, the comparison of bacterial DNA profiles under various storage conditions. Based on the experimental results, guidelines were drawn They detaile access to soil sampling in scenes and agents, including vehicles, shoes, spades etc, their collection, handling, and storage.
A multi-laboratory evaluation of a mock-crime scene using various analytical and geographic localization techniques, most showing the ability to compare a questioned soil to a particular crime site was designed, created, sampled and analysed. The scheme included 12 partners from MiSAFE and outside MiSAFE. It showed the ability of soil forensics analyses to correctly define the relationship between soil samples.

Accessing and sampling soil
In conjunction with the statistical expert partners, BioSS, and in close consultation with police practitioners and forensic analysts, it has produced a “soil sampling kit” (Fig. 3) – a template guiding the sampling of soil in forensics investigations.

Storing soil samples

Soil samples were collected and subjected to various storage conditions. Three contrasting soils were evaluated: peaty gley, podzol, and brown earth and a range of different storage treatments were tested:
Exp1: dry winter scenario or cool/dry storage (open container with desiccant at 5°C),
Exp2: standard laboratory storage condition (sealed containers, at 5°C),
Exp3: Air dried at ambient temperature,
Exp4: Air dried at ambient temperature with regular rewetting (at 20% residual water content),
Incubation up to 50 days, sampling at 5 staggered time points.
The DNA was subsequently extracted with the ‘Hutton’ protocol (TM1, WP1), and using kits K03 and K0. The data were then evaluated using T-RFLP analysis. Results on the effect of storage conditions on T-RFLP analysis showed that there was clear separation of the three soil types, independent of the storage conditions (Fig.4).

Further analysis was performed on a per soil basis:
The brown earth soil, the humus podzol and less distinctly the peaty gley soil showed that all treatments clustered together, with the exception of the wetting and drying treatment. This important finding suggests that samples should not be rewetted if dried and then subsequently analyzed.
Conclusion: rewetting exerted the strongest influence on the soil microbial community and resultant T-RFLP data. However, the result can be seen to be dependent upon soil type. For microbial analyses, soil samples should be transported to the forensic laboratory as quickly as possible to minimise any influence of wetting or drying that may ensue.

A survey of soil forensics practice in Europe
An on-line digital survey was carried out among forensic practitioners to assess the current situation in Europe regarding the current use and future possible use of microbial community analysis for forensic applications. A response from over 30 was received. This was to ascertain the current situation in Europe in the use of microbial fingerprints in forensic work (Fig. 5).
The most common approaches are directed towards microbial 16S rRNA genes, with the most commonly mentioned molecular methods being Roche 454, Illlumina, ABI Fast MicroSeq 500, or T-RFLP. For the exhibit case work soils, 50% were currently stored in plastic bags/jars, 38 % in paper bags and 10% in glass jars. For the control soil samples, 32% were stored in glass containers, 55% in a plastic bags/jars and 10% stored in paper bags/envelopes.

Conclusion: Microbial analysis is clearly underused as a forensic tool in Europe.


An inter-laboratory mock crime scene test
A mock crime was designed, based on a real case example, with a scenario that a local farmer discovers an area of disturbed soil on his land, and subsequently finds a packet of drugs buried underground. Three locations were chosen to use in the collaborative laboratory assessment exercise that included locations with whether similar or different characteristics to the crime scene. These sites were close to where the original mock crime scene experiment was set up, to provide a larger database to assist in interpretation of the resultant data sets.
Twelve participating laboratories from MiSAFE and outside MiSAFE received samples provided by the James Hutton Institute and analysed them using their own case approaches to enable comparisons to be made on the discrimination between soil types and to enable a test of the approaches used.
Samples were collected, recorded, stored and transported according the sampling guidelines developed in the MiSAFE project and shipped to participants fresh, frozen or air-dried, as requested by the individual correspondents. Some of the items (spades) included a mixture of soils. The methods of analysis were categorized as “biological”, “chemical”, “colour”, “mineralogy”, “palynology” and “morphology”.
Three main questions were asked of the participating laboratories:
Can you describe what type of environment this soil might have come from?
Could any of the three sites be related to the location of the soil on either Spade? A or B?
a) In your professional opinion, could the soil from the Spades A or B have come from any of the three control sites?
b) Can an evidential value also be provided in the statement as to whether either of the samples from the spades could have come from any of the three control sites?


Biological (DNA-based) methods were the most in matching items to site when soils were not mixed. The poorest outcome was from the use of pollen. Overall, only one participant (an experienced practitioner outside of the MiSAFE group) was able to answer all of the questions correctly.
Conclusion: Soil DNA-based methods worked satisfactorily for relating an unknown soil to a particular site. Where there is a mixture of soil on the questioned items DNA-based methods were not so efficient at the moment. Laboratories in case work would and should use a combination of methodological approaches and thus are more likely to come to the correct interpretative conclusion if complementary approaches are used. Soil DNA methods show promise as being one of the complementary approaches to be adopted.

Guidelines
Detailed guidelines for field, laboratory and storage procedures were drawn up. The guidelines have a common core, and local adaptions to the specificities of the subject. The guidelines include:
Field sampling
Sampling from questioned items-shoes and spades
Sampling from questioned vehicles-car
Sample storage
Recommendations were drawn up based on the information concerning potential implementation of a soil DNA forensic approach obtained from an international digital survey, and from testing of a range of storage conditions and of the sampling kit, presented in the first report.

For each of these items, the following sections are detailed:
Considerations: considerations are item specific; they describe additional parameters that may be useful, in addition to the methods forming the basis of MiSAFE; behaviors to adopt vis-à-vis the item; and a range of dos and don’ts as guidelines.
Health and Safety considerations.
Minimum equipment: describes the items which should be brought to the field for accessing, sampling, describing, transporting, and storing samples.
Sampling protocol: specifies how to examine, record, decide upon the site/location/position (depending on the size of the sampled item from field size to shoe), sample size, tools to employ, tools to store and guidelines to store samples, conditions of transfer to the laboratory, and actions to be taken post-sampling for medium to longer term storage.
Storage.

WP03: Evaluation of novel technologies for soil forensic use

Involved partners 01 HUJI, 02 LIB, 04 ECL, 05 JHI
Objectives
Determine the best biomarker-technology combination for the Park grass reference soil
Validation of biomarker-technology combination with other soils
Identify the best performing and robust DNA extraction methods based on the extent and reproducibility of revealed microbial diversity
Work in this WP will assess the appropriateness of novel environmental molecular technologies to forensics frameworks, considering the problem of the presence of a ‘rare biosphere’ in every soil and bias relating to sampling, sample size, sample types, and DNA extraction protocols.

Main results and achievements
WP3 has performed the following tasks and reached the following achievements:
The different fingerprinting technologies: RISA and phylogenetic microarray, and gene sequencing were evaluated for soil microbial analysis and marker efficiency. This showed that microarrays are not efficient for soil forensics but that RISA successfully differentiates between soil types. The 16S rRNA gene marker is the most efficient while other house-keeping genes, and antibiotic resistance genes do not provide enough spatial resolution to be useful in soil forensics.
The impact of DNA extraction methods on realized soil microbial diversity was investigated and it was shown that extraction has a significant effect of community structure.

New biomarker – technologies combination
An evaluation of the rpoB and rpoC genes, including sequencing of cloned sequences from soil sample, and QPCR of selected antibiotic resistance genes was performed against the standard 16S rRNA gene marker. The alternative genes were not specific to a given soil and thus cannot be used in forensic science to discriminate between soil sample origins. This approach was not further used.

Evaluation of RISA under different conditions
Application of RISA: evaluation of sampling time and cold storage.
The Ribosomal Intergenic Space Analysis (RISA) was used to evaluate the effect of sampling at different time periods and the effect of storage. We concluded that temporal sampling and cold storage did not affect significantly bacterial communities according to RISA technology. However a cluster analysis restricted to the 3 grouped soil samples could separate them, but with a very small distance between samples (Fig.6).
A new series of statistical analysis was performed without the agricultural samples Apple. Looking in details to statistics allowed us to see that the 2 first axes did not discriminate among sampling time, but that samples could be separated according to the year of sampling when we considered the first and the third axis of the PCA and when we used the ward clustering method with ordiclust (see the Cluster Dendrogram, Monte Carlo Test p-value: 0.001).
Conclusion.. Storage (according to conditions described in WP2) does not affect bacterial community structure. Sampling time had little effect as well – importantly these are soils from Rothamsted in the UK. Seasonal effects were much more marked in Israel (see WP4).

Comparison between RISA and phylogenetic microarrays.
The RISA approach demonstrated that the bacterial diversity from sample S34 (Sand sample Group 3 extraction 4) was statistically different from others according to the clustering analysis. Other Sand samples clustered together but could not be easily differentiated according to sampling distance. The Terra Rossa and Rendzina samples were separated with the clustering analysis. Groups 1 and 2 (25 and 75 m) could not be differentiated but Group 3 (100 m) for Terra Rossa and Rendzina samples clustered apart (figure on the left). RISA differentiated between 3 Israeli soil samples and could separate in 2 cases 100m-samples from 0- and 25m-samples, but the 0m and 25m samples were not differentiated with this data treatment approach.
The phylogenetic microarray approach (Fig. 7) demonstrated that the centroids of samples from Terra Rossa grouped together, as those from Sand, whereas those from Rendzina are more dispersed. As concluded in the previous report, phylogenetic microarrays did not completely differentiate the samples according to their origin but provide some evidence of similarity.
As part of the mock-crime scene partnership, analyses were performed within the frame of WP3, using RISA, microarrays and Illumina MiSeq high throughput sequencing. The results obtained matched those described above in the WP4: RISA and MiSeq are effective methods for soil spatial comparison – microarrays are not.

Conclusion. RISA is an effective methods for distinguishing between soil type. Phylogenetic microarrays do not provide the needed spatial resolution.

WP04: Delimiting spatial and temporal boundaries of microbial soil profiling

Involved partners 01HUJI, 02 LIB, 03 CLCBio, 04 ECL, 05 JHI, 06MOPS/INP, 07GC
Objectives
Define the spatial resolution of MPS and T-RFLP under various soil conditions and sampling distances
Develop and test a sample manipulation procedure to address seasonal effects of sampling
Address legal issues of manipulating samples

Main results and achievements
The results and achievements of WP4 include:
A comparative analysis of PCR protocols, primer tags, DNA dilution effects, PCR replicates, and TRFLP runs on the resolution of soil samples resulting in an optimized protocol for soil DNA analysis by TRFLP.
A comparative analysis of 18 statistical pipelines for analysis of TRFLP data, yielding a protocol for TRFLP data handling and statistical analysis.
A comparative analysis of marker genes between 3 closely related soils and within soils (distance-decay), confirming that the 16S rRNA gene is a most reliable and precise marker.
A comparative analysis of methodological approaches, including chemical, microarray, pattern-based, and sequencing approaches, and alternative marker, applied to soil sample discrimination. This showed that all methods besides the array-based method and the alternative marker significantly distinguished between soil samples some with high spatial resolution.
An analysis of a soils under seasonal changes and a manipulation experiment trying to re-created seasonal changes. It showed that although the potential exists, further research is required to mitigate seasonal fluctuations for matching samples retrieved at different times.
A scrutiny of legal aspects on soil DNA uses.

TRFLP optimalization
TRFLP is readily applicable in forensics laboratory as it is uses the same technological platforms widely used in human DNA analysis. Therefore an effort was made to optimize its performance of soil forensics analysis.
Six parameters were tested in an attempt to increase the efficiency of 16S rRNA TRFLP: choice of primer pair; single forward, single reverse, double fluorescence tagging of the PCR amplicon; concentrating DNA afterTaqI digestion; number of PCR amplifications per sample; number of capillary electrophoresis runs per PCR amplification; and choice of statistical analysis procedure. The 341F/907R primer pair displayed the highest DNA yield as well as the highest number of terminal restriction fragments (TRFs); Double-tagged primers and concentrating DNA produced a high number of TRFs combined with high between-soil dissimilarity and within-soil similarity; any single capillary electrophoresis (CE) run captured as little as 50% of the bacterial diversity (as represented by the number of TRFs), with a logarithmic increase in the number of unique fragments as the number of CE runs increases (Fig. 8). This increase in sampling effort (from 1 to 5 to 10 runs) caused the differences between soil types to remain the same while the differences between replicates from the same soil decreased substantially (Fig. 8). An optimized data handling routine was also developed, based on peak score, excluding raw data, but including power transformation.

Comparative analysis of fingerprinting technologies for bacterial soil forensics
This section is the culmination of a multi –WP and partner effort coordinated by WP4, that lasted over the two years of MiSAFE. Various DNA-based technologies that are commonly employed for studying the structure of bacterial communities were compared using different soils as tests. The aim was to provide the forensic practitioner with a microbial genetic profiling tool that will bring soil into routine forensic investigation and judicial proceedings. Accordingly, the technologies should be applicable at the forensics lab level, be robust, and provide high resolution. This is made possible by capitalizing upon the great advancements in DNA technologies and computing and develop these into tools for crime-fighting and crime-prevention.
The soils used in this study are the same renzina, terra rossa and sand soils used in many investigations in MiSAFE. The technologies tested included RISA, TRFLP with the rpoB marker, TRFLP with the 16S rDNA marker, with 1 or 3 runs, and high throughput sequencing of the 16S rDNA marker (Roche 454, Illumina MiSeq, PGM Ion Torrent). In addition an organic chemistry method, n-alkane (long chain hydrocarbons) of plant material was included. The best discrimination was achieved with n-alkanes, RISA, TRFLP with three capillary runs and NGS Miseq. However, it should be noted that all approaches, except the phylochip and the TRFLP-rpoB differentiated between the soils with high significance (Fig. 9). Further, the size of the differences between the soils were in accordance with the soil types, i.e. renzina and terra rosa were less different than any of these two to sand (Fig. 9).
Conclusion. Significant differences between soils can be detected using independent methods, bringing robustness to the analysis.

Temporal effects on soil samples and their mitigation
Seasons, through the change in temperature, precipitations and light have a great impact on the environment, including soil microbial life. It is therefore of prime importance for soil forensics to describe this changes and eventually to develop approaches to mitigate them, as soil samples to be compared in a forensics case may not all be retrieved at the same time. WP4 repetitively sampled and analysed the same sites, in a variety of soils, throughout the year. In addition, WP4 attempted to devise an experimental protocol for reducing these seasonal effects, thereby increasing soils’ efficiency as forensic evidence.
The three Gimzo (Israel) soils as above were used in this study and were sampled in summer 2013 and in winter 2013/2014. There were analysed directly, and winter samples were further used in microcosm experiments. These experiments aimed at comparing incubated and field samples from different seasons and to explore the possibility of re-creating seasonal fluctuations experienced by soils. Soils were thus incubated in pots, in a phytotron. This included: retrieving samples from the field in winter; incubating them in pots in for 16 days at 10°C after wetting; drying them at 28C for 7 days; rewetting at 10C for 14 days. Pots were sampled at the beginning and at the end of the experiment and at days 2, 3, 5, and 7 during drying. Samples were processed and analysed by TRFLP as above.
The three soils were separated with significance notwithstanding seasonal although the seasonal effect was very strong as the different soils clustered more closely together based on season that on soil type. During incubation of the soil pots, the bacterial communities shifted as the soils dried. The communities converged in sand but in terra rossa and renzina (heavier soils) the communities became more similar but were still significantly different, indicating that they had not yet stabilized. Thus the drying process alters the community of the sampled soils towards another (dynamic) equilibrium.
This mitigation experiment of seasonal effect, i.e. the wetting of hot dry soil mimicking summer to winter-like conditions did not match the field samples retrieved in summer and in winter (Fig. 10). Strikingly, the differences between the seasons were more pronounced than the differences between soils. However, partial cycling was observed: while the wetted and rewetted sand and renzina soils clustered according to treatment, in terra rossa the wetted and rewetted soils were mixed. This indicates that their communities converged (Fig. 11).

Conclusion. Mitigation as performed here could not re-create the natural conditions. However, it may be possible to manipulate soils, as shown by the partial success in the cycling part of the experiment.

WP05: Bioinformatic tools

Involved partners 01HUJI, 03CLCBio, 04ECL, 05JHI
Objectives
This work package addresses the bioinformatics needs of the project and has three main objectives:
To build an integrated state-of-the-art solution for data handling, quality control, analysis, and visualization of MPS data from soil-samples
To establish a standard analysis pipeline for T-RFLP data: data handling, quality control, soil sample similarity analysis, and visualization
To store, structure and query the soil data (physical characteristics, environmental parameters, T-RFLP and MPS) in a geo-forensics database.
Main results and achievements
The results and achievements of WP5 are:
A fully-fledge, inclusive, user-friendly and CPU-efficient metagenomics workbench that is readily applicable to forensics investigations. It includes features for high throughput genomics work as well as a platform for TRFLP analysis. The software package is acommercialized, It fully integrates data and statistical analyses and visualization; a guidance for a biogeoforensics database.
A website for the software is to be found at: http://www.clcbio.com/products/clc-genomics-workbench/

Geoforensics database
The data structure is made to conform to the genetic metadata listing outlined by the Genomic Standards Consortium (GSC). The geoforensic database is a small-scale prototype to explore the potential for a Pan-European database. For the software functions key requirements were identified as: location – query soil and vegetation at a location; display background data for context; overlay of field observations; combine different types of data (graphical, analytical); query attributes in database(s); statistical analysis of feature attributes (e.g. correlations between soil attributes in sample with those in a database); exporting data in to a statistical package; production of maps as outputs in reports/ evidence. The selected framework for the spatial database and its use is the ESRI ArcGISpackage (www.esri.com) which has global use, the required functionality, capabilities for import and export of data and high quality tools for creating cartographic outputs.
The spatial aspects of the geoforensic database are informed by the development of standards for a European soils database in the GS-Soil project. For the data specifications, MiSAFE builds on the eContentplus of GS Soil and its reporting on best-practice for aligning European soil database schemas to INSPIRE. ‘Reference samples’ are accessible to external users, but that ‘evidence samples’ (i.e. relating to a specific judiciary process) can have restricted access. The deposition of data can be restricted to forensic units and authorized bodies. For a source of backdrop data for referencing field observations, interpretation in intelligence for reduces areas of search, and cartographic map output, the Openstreetmap has been identified as the standard to be used. This provides standardisation across Europe in terms of data capture methods, attributes and cartographic representation, thus overcoming some significant cross-border differences, which would otherwise limit the pan-European scope of the implementation.
Conclusion. Specifications have been developed, and the final output is a prototype database with spatial and aspatial attributes and extensive software capabilities.


WP06: Evaluation and validation of protocols and material, legal support

Involved partners 01HUJI, 02LIB, 05JHI, 06MOPS/INP, 07GC
Objectives
To validate of Best Practice and Standard Operating Procedures
To validate the testing procedure for forensics scenarios
To validate the analytical software, validation of the structure and use of the geoforensic database
To undertake a legal review of soil sampling, storage, processing and analytic procedures and of data storage, access and use.
Main results and achievements
WP6’results and achievements are:

Evaluation and validation of protocols and material, legal support ; Evaluation and validation of tools previously proposed by MiSAFE partners for soil bacterial community analysis for forensic scenarios within an ISO frame.
Legal considerations of use of tools for soil bacterial community analysis (EU Comparative Law)

The following protocols were evaluated:
Protocols for soils sampling and handling (ISO 17020)
Protocols for use of forensic scenarios as corroborative tests (ISO 17025). This included the validation of a TRFLP protocol.
Reports on legal aspects of microbial fingerprints.

Potential impact and main dissemination activities and exploitation results

MiSAFE has:

Developed a practical and multi-parametric model for evaluating DNA extraction protocols and products. This approach has yielded a set of methodologies and designated products for forensic work.
Demonstrated, on a large number of soils, under various climates and seasons and using multiple independent technologies that soil bacterial communities provide a powerful tool for obtaining forensically relevant information with high spatial resolution and robustness.
Produced guidelines and protocols, and their validation for soil microbial sampling, handling, storage and analysis in forensic contexts.
Defined the legal backgrounds under which soil microbial forensics can be applied.
Exposed forensics laboratories and practitioners to the use of microbial soil forensics and engaged non-MiSAFE laboratories in soil analysis.
Developed map-based tools for the geographically sorting of crime-related scenes.
Developed, implemented and launch a fully-fledged commercial software platform for the analysis of microbial communities in soil samples and in other environments.

The main impact of MiSAFE is that is has shown that “soils can be brought to Justice”. The implications are that the forensic labs can add another tool to their investigation tool-box (Fig. 12).

Figure 12 depicts the full chain of events from the site to the court as may happen to a soil sample. Each of the various steps was evaluated, tested, and implemented successfully.

Soil investigations are not limited to personal crimes. Soil bacterial profiling may be applied to help decipher environmental crimes; to link agricultural performance to soil biological properties; to map sites for metagenomics analyses, and more.

Dissemination

Dissemination of the outputs of the MiSAFE research was carried out under WP8. A website was established (Fig. 13) and there were over 60 related events (talks, publications, interviews for example). The international impact was considerable with successful collaboration with European and global networks. The details of the dissemination section are provided below.

Potential Impact:
MiSAFE has:

1. Developed a practical and multi-parametric model for evaluating DNA extraction protocols and products. This approach has yielded a set of methodologies and designated products for forensic work.
2. Demonstrated, on a large number of soils, under various climates and seasons and using multiple independent technologies that soil bacterial communities provide a powerful tool for obtaining forensically relevant information with high spatial resolution and robustness.
3. Produced guidelines and protocols, and their validation for soil microbial sampling, handling, storage and analysis in forensic contexts.
4. Defined the legal backgrounds under which soil microbial forensics can be applied.
5. Exposed forensics laboratories and practitioners to the use of microbial soil forensics and engaged non-MiSAFE laboratories in soil analysis.
6. Developed map-based tools for the geographically sorting of crime-related scenes.
7. Developed, implemented and launch a fully-fledged commercial software platform for the analysis of microbial communities in soil samples and in other environments.

The main impact of MiSAFE is that is has shown t “soils can be brought to Justice”. The implications are that the forensic labs can add another tool to their investigation tool-box (Fig. 12).


Figure 12 depicts the full chain of events from the site to the court as may happen to a soil sample. Each of the various steps was evaluated, tested, and implemented successfully.

Soil investigations are not limited to personal crimes. Soil bacterial profiling may be applied to help decipher environmental crimes; to link agricultural performance to soil biological properties; to map sites for metagenomics analyses, and more.

Dissemination

Dissemination of the outputs of the MiSAFE research was carried out under WP8. A website was established (Fig. 13) and there were over 60 related events (talks, publications, interviews for example). The international impact was considerable with successful collaboration with European and global networks. The details of the dissemination section are provided in the tables below.


List of Websites:
http://forensicmisafe.wix.com/misafe

Coordinator:
Prof. Edouard Jurkevitch
edouard;jurkevitch@mail.huji.ac.il

Informations connexes

Contact

Jane Turner, (Coordinator for Europe)
Tél.: +972 2 6586676
Fax: +972 72 2447007
E-mail
Numéro d'enregistrement: 184018 / Dernière mise à jour le: 2016-06-03
Source d'information: SESAM