Objectives

The exemplar projects will serve as a model of integrating forensic research in Europe. The work progresses from sample collection at the crime-scene to genotyping, data analysis and the preparation of custom marker multiplexes to address a specific investigative need. These main activities provide the focus for other important parts of the overall project, such as dissemination activities. At all levels, each exemplar is overseen and progress is monitored by the project management team. The overall strategy is summarised as follows: use existing data and expertise from the project partners to develop the ?next generation? of integrated DNA profiling methodologies to maximize the chances of identifying perpetrators of crime in major investigations.

In a second step a call for competitive research funded projects in forensic genetics will be open to the scientific community selected on the basis of criteria such as being multidisciplinary or their contribution to the solution of practical problems in forensic genetic casework. This will allow the identification of new partners and to extend the network towards the creation of a European virtual centre of excellence, which is the main aim of WP2.

The overall objective of WP3 is driven by the need to rapidly address a number of scientific and practical challenges slowing down the practical applications of forensic DNA analysis. Therefore, within the first 30 months of the network three collaborative exemplar projects (EPs) will be carried out by EUROFORGEN-NoE core members. They are aimed to develop scientific solutions for the practical problems described in the EPs, and they serve to exhibit the international collaborations required to enhance the research landscape in Europe. The subsequently described three EPs will be complemented by a competitive call for research proposals after this first period of active research (see WP1) to identify and integrate new research ideas into EUROFORGEN-NoE.

EP1 - Crime scene investigation and human DNA discovery

The objectives of exemplar project 1 are:

• Evaluation of the usefulness of rapid DNA detection, mRNA, miRNA and epigenetic markers, as well as development of a test for the detection of human DNA at crime scenes.
• A protocol to describe best practice for discovering and handling trace DNA evidence originating from crime scenes

Description of Work

Task 1: Development of new tests to identify body fluids/tissues

As DNA profiling evidence becomes more sensitive, it is even more important to be able to provide information to the investigator about the cellular origin of the DNA. Presumptive tests have limitations regarding the cell types that can be assessed and in their specificity and sensitivity. Recently, new RNA profiling multiplexes have been developed that are able to distinguish between blood, vaginal mucosa, semen, menstrual secretion, skin and saliva. Because RNA can be co-extracted with DNA no additional consumption of precious stain sample is required for the identification of the biological sample. The purpose of this package will be to evaluate the robustness of existing markers; to develop new tests to identify additional markers for other body tissues utilising both mRNA and micro RNA markers. Epigenetic (methylation) markers will be explored as an adjunct.

In addition to clearly visible biological stains such as e.g. from blood, there are DNA traces invisible to the human eye. Nevertheless, such traces may relevant providing valuable information about persons present at the crime scene. To detect such traces carrying human cells (and DNA), it will be attempted to develop a rapid search test indicating the presence of human DNA.

The concurrent rapid development of portable micro-PCR instruments may enable the simultaneous analysis of human RNA and DNA at the crime scene. Simultaneous analysis will offer the authorities real time answers on the two basic questions in forensic science – What (is the biological substance involved) and Who (left the DNA).

Task 2:  A Best practice protocol

Much of work package 3 is aimed towards improving the quality of information that can be provided to investigators. The development of new body fluid/tissue markers in WP3/EP1 is complemented by WP3/EP2 (guiding investigations by genetic analysis of physical traits). Consequently, a whole range of new tests will be developed that will completely change the way that we process cases. From the crime-scene to the final report there will be many more decision points to make about which tests to utilise and this will in turn depend upon the circumstances and context of the case in question. The second task will be devoted to marshalling the information in order to provide a document that outlines best practice at the crime-scene so that any laboratory analysis can be focussed to answer questions that are the most relevant to the investigation. The information compiled in this task will form the basis of educational workshops envisaged under WP5.

EP2 - Guiding investigations by genetic analysis of physical traits and tailored multiplex development

The objectives of exemplar project 2 are:

• The selection of SNP panels to optimize information content to establish identity
• To optimize development of small scale multiplexes:
• A broad brush approach for general-purpose identification e.g. continental ancestry
• A fine-tuned approach for a specific major crime event resembling the Madrid bombing
• To optimize whole genome scans of low template DNA
• To evaluate use of new medium-throughput genotyping platforms e.g. Taqman OpenArray
• To develop methods to rapidly incorporate SNPs linked to physical traits into new tests

Description of Work

This work-package will identify candidate genes as associated markers for physical characteristics to identify ancestry, pigmentation, etc. and will carry through the rapid preparation and evaluation of large multiplexes. This will identify the key candidate gene variants for incorporation into trait-informative multiplexes. The output will be tailored to the investigative needs to include: identification of DNA components from contact traces, use of genetic ancestry markers, use of physical trait analysis markers (based in part on identified ancestries) and use of Y-chromosome surname predictive markers. The data exchange framework between laboratory and police investigators would centre on high density SNP array data to inform production of specific marker selections for smaller-scale multiplexes that can address common eye-witness queries such as ancestry, eye colour and age or can focus on specific investigation leads. So the main technological challenges comprise: adaptation of whole genome scan systems to analyze contact trace DNA, development of bioinformatics systems to rapidly identify SNPs most useful for individual and family-based identity plus ancestry and trait analysis, and collation of data from studies finding the strongest associations of SNPs to physical characteristic traits. In large part the genotyping technology is mature and widely established, particularly high-density arrays alongside specific SNP tests of proven forensic sensitivity.

EP3: Bioinformatics in silico modelling and statistics

The objectives of exemplar project 3 are:

• To carry out in silico simulations to determine the best strategies to identify perpetrators at major crime scenes.
• To optimise a strategy to provide intelligence to investigators when STR analysis fails to find suspect(s).
• To measure effectiveness of customized multiplex systems to determine surname, ancestry, age etc.
• To provide modules for complex mixture determinations
  

Description of Work

There is an obvious need for bioinformatics and statistics whenever complex models and large amounts of data are involved. In silico is an expression used to mean ‘performed on computer’ or via ‘computer simulation’. For this project, in silico models and experiments supplement traditional models and data sources. In brief, the objective of this package is two fold:

• To perform relevant analyses without access to case data. This is the in silico objective (1) above.
• To develop and facilitate methods of statistics and bioinformatics to be used to solve the problems described in objectives 2, 3 and 4 above.

Task 1: In-silico simulation studies to optimise investigative strategies

WP3/EP3 complements WP3/EP2 to facilitate gene discovery. Chips with 500,000 SNPs or more are routinely used to discover genetic causes for disease. Data analyses based on such great amount of data is complex. However, this is greatly facilitated by freely available software available both for simulation and analyses.

The following questions will be addressed:

• What are the optimal genetic markers to use for different purposes?
• How many markers are needed to obtain reliable answers?
• What is the robustness of the overall strategy to be employed?

The in silico experiments can be designed using traditional methods of statistical design and the output can be analysed using standard statistical methods. The purpose will be to determine robustness and effectiveness of the multiplexes provided by WP3/EP2 prior to implementation.

Task 2: Complex Mixture Interpretation

The WP3/EP3 workpackage will adopt an open-source software package ‘Forensim’ (http://forensim.r-forge.r-project.org/) developed by Hinda Haned (NFI). We will support and complement the work that has already been undertaken to develop mixture interpretation modules as follows:

• Develop methods to interpret and to deconvolve STR and SNP mixtures incorporating concepts of drop-out and drop-in.
• Carry out comparative studies with existing and interpretation methods based on alternative software solutions
• Devise methods to test robustness of proposed solutions
• Support validation exercises
• Support casework labs in pilot studies
• Contribute to workshops under WP5.

EP4: Association of a Body Fluid with a DNA Profile by Targeted RNA and DNA Deep Sequencing

The objectives of exemplar project 4 are:

• To set up a targeted mRNA/miRNA next generation sequencing approach for body fluid/tissue identification and to establish a probabilistic approach to call/predict the presence of a body fluid.
• To select a set of SNPs for each body fluid/tissue and ethnicity, that discriminates individuals the most. Specific assays will be developed for DNA and RNA biomarkers.
• To combine the RNA analysis with gDNA STR sequencing, allowing simultaneous forensic tissue identification and human individual identification.

Description of Work

In WP3/EP4 a targeted mRNA/miRNA next generation sequencing approach for body fluid/tissue identification will be set up. This includes the evaluation of a suitable library preparation protocol and sequencing platform, as well as the selection of the most suitable markers, and a bioinformatics pipeline for processing the raw data. A probabilistic approach will be established to call/predict the presence of a body fluid.

Based on the final mRNA/miRNA markers a set of SNPs will be selected, for each body fluid/tissue and ethnicity, that discriminates individuals the most. Specific assays will be developed for DNA and RNA biomarkers. With these results a body fluid can be assigned to a specific individual. This will be especially interesting for mixed body fluid samples, to find out who contributed which body fluid.

Combining the RNA analysis with gDNA STR sequencing will allow simultaneous body fluid/tissue identification and human individual identification. With NGS data more quantitative results are generated which are more suitable to be used in probabilistic approaches to infer genotypes and their corresponding cell type in mixed donor samples.

A final goal would be to have a ready-to-use NGS solution for DNA and RNA analyses of casework samples and to develop interpretation guidelines for DNA and RNA results that follow forensic standards and can be used in court.

EP5: Development of innovative electrochemical biosensor technologies for the detection of tissue specific DNA methylation

The objectives of exemplar project 5 are:

• Identification of 5-10 candidate tDMRs for four body fluids (saliva, semen, blood, menstrual fluid)
• Development of an ultrasensitive ECL biosensor technology for methylation detection with otpimisedsignal-to-noise ratio and shifting of luminescent emission beyond the absorption range of whole blood for tDMRrecognition without the need for extraction or pre-concentration
• Performing full forensic validation of the ECL based tDMR detector arrays with determination of the limit ofdetection and comparison with existing methods for body fluid identification

Description of Work

The aim of our research is to further our knowledge on tissue specific differentially methylated regions (tDMRs) within the human genome and to implement an entirely new technique into forensic science, namely electrochemoluminescence (ECL) based biosensor arrays. During the initial phase of the project we will identify suitable candidate tDMRs for the identification of the four body fluids saliva, semen, menstrual blood and blood. These markers will then be used for the development of the novel ECL biosensor.

We seek to exploit ECL labelling based on standard labels, such as ruthenium complexes, as well as new materials with near-infrared (NIR) properties, such as quantum dots and novel organic complexes to develop an ultrasensitive method for the detecting and monitoring of differentially methylated DNA sequences.

The outcome of this work will be a portable biosensor array for the simultaneous identification of four body fluids, namely saliva, semen, blood and menstrual fluid, in extracted DNA as well as directly in forensically relevant body fluids without the need for prior treatment or extraction.

This is particularly challenging for body fluid mixtures containing whole blood due to the absorption characteristics of blood. To overcome this challenge, we propose to use novel near infrared (NIR) ECL complexes which shift the standard peak emission wavelength into the blood transmission window to allow specific detection of light signals.

EP6: Forensic DNA phenotyping of hair structure for investigative purposes

The objectives of exemplar project 6 are:

• Generating a set of most informative DNA predictors for human hair structure from newly and previously identified genetic determinants.
• Establishing and validating a statistical model for hair structure prediction from DNA data.
• Developing and forensically validating a multiplex genotyping assay for analyzing the identified DNA predictors of hair structure.

Description of Work

With WP3/EP6 we aim to extend the concept of Forensic DNA Phenotyping (FDP), i.e., the DNA description of externally visible characteristics to provide investigative leads for finding unknown perpetrators unidentifiable via conventional DNA profiling, to hair structure.

We will start with identifying new genetic determinants of hair structure i.e., weather the head hair is straight, wavy, or curly via large scale genome-wide association studies (GWASs) in multiple population cohorts involving thousands of Europeans in collaboration with partners from the International Visible Trait Genetics (VisiGen) Consortium. From the statistically significant outcomes, DNA predictors will be selected, and a statistical prediction model will be built and tested in independent samples. A multiplex genotyping assay will be developed and forensically validated based on technologies suitable for forensic DNA analysis, and applied to independent samples for further validation of the prediction markers and model in collaboration with EUROFORGEN-NoE partners.

The final goal of this project, provided that the research outcomes allow, is a DNA test system for establishing hair structure phenotypes from DNA samples such as those found at crime scenes that includes a forensically validated multiplex genotyping assay and a validated statistical prediction model. We envision that the delivered DNA test system for hair structure will be combined with previously developed DNA test systems for eye, hair, and skin color, perhaps hair loss, and age for practical FDP purposes, allowing to better describe the appearance of unknown persons involved in crime cases or missing person cases to help identifying them.