Assigning weight of DNA evidence using a continuous model that takes into account stutter and dropout

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Abstract

achieved full profiles from 70% of samples analysed. Sensitivity experiments were conducted using liquid blood aliquots which were serially diluted to achieve dilutions between 0.1 ng/µl and 0.0007 ng/µl. From eight dilutions, 5 µl aliquots were simultaneously sampled for the automated and manual DNA profiling systems. The results of both systems were assessed using allele scoring as per NDNAD loading criteria. The first sensitivity experiment (n = 3) showed 69% of samples gave equal to or more loadable alleles when profiled using the automated profiling system. The second sensitivity experiment (n=5) showed 39 % of samples gave equal to or more loadable alleles when profiled using the automated profiling system. As the results from the sensitivity experiments are variable we will maintain a manual profiling system for critical samples or samples thought to yield-low concentrations of DNA. Assessing the audit trail for samples showed that on all occasions the sample name was correctly transferred from the extraction batch to the quantification record, PCR record, and DNA electropherogram printout. In conclusion, we have designed a system which can obtain DNA profiles and manage sample data effectively from all the tested sample types such that we can process the majority of samples encountered within SPSA Forensic Services, Glasgow.

doi:10.1016/j.scijus.2009.11.071

A criminalistic approach to biological evidence: Trace DNA and volume crime J.J. Raymonda,b, R.A. van Oorschotc, S.J. Walshd, P.R. Gunna, C. Rouxb a NSW Police Force, Forensic Services Group, Sydney, Australia b University of Technology Sydney, Centre for Forensic Science, NSW, Australia c Victoria Police, Forensic Services Centre, VIC, Australia d Australian Federal Police, Forensic and Technical Services, Canberra, Australia In the early days of forensic DNA analysis, the technology's sensitivity was limited to biological stains such as blood and semen, and issues relating to the transfer of such biological substances not a major concern. However with technological developments it is now possible to detect trace DNA from handled objects, and therefore broader principles of criminalistics have become more relevant and can now be applied to biological evidence. However, as DNA dips into the realm of trace evidence, the interpretation becomes much less straightforward. The contested issue in legal argument over DNA evidence is now less often the identification of the sample, but rather issues such as transfer, persistence, and exactly how that sample came to be present at the crime scene. This research investigated the trace evidence characteristics of DNA to better understand the behaviour of trace DNA, with the aim to provide a Bayesian interpretation of this evidence. Given the unlimited number of variables affecting the recovery of trace DNA, two volume crime types, burglary and robbery, were chosen on which to base the research. The trace evidence characteristics of abundance, transfer, and persistence were investigated in relation to these crimes. Background levels of DNA on residential burglary entry points and items commonly stolen during a robbery were assessed and were found to be significant, in some cases particularly if low copy number techniques are employed. Small amounts of ‘non-owner’ or ‘non-resident’ DNA were found on the surfaces, and therefore it cannot be assumed that DNA foreign to the victim must belong to the offender. The level of DNA transferred during these offences was found to be lower than the level of background DNA present in several instances. The amount of DNA retrieved after being deposited onto various surfaces was found to decline rapidly when in outdoor environments; however, full profiles were recovered from samples left inside at room temperature after six

weeks. A major criticism of Bayesian reporting of evidence is the lack of available data to be used in the model. With studies like those described here to provide such data, a more robust interpretive framework may be developed for trace DNA. doi:10.1016/j.scijus.2009.11.072

The definitive identification of body fluids using mRNA R.I. Fleming, A.L. Bowden, W. Hermiz, J.S. Buckleton, S. Harbison Institute of Environmental Science and Research Ltd, Auckland, New Zealand Biological stains from blood, semen, and saliva, or mixed body fluids are commonly encountered in forensic casework. When mixtures of body fluids are present, it can be difficult to identify the minor component using traditional methods. Current DNA profiling methodology can yield a DNA profile from a sample of body fluid, but the emphasis for forensic scientists now is not on from whom did the DNA come from, but what body fluid or cell type did this DNA profile originate. There are many circumstances where it is important to know not only who the sample came from but also what body fluid the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) to identify blood, saliva, semen, menstrual blood, and vaginal secretions. Messenger RNA transcripts that are specific to each type of body fluid have been identified, primers designed, and a multiplex PCR system developed to identify the body fluid(s). This multiplex can detect semen with and without spermatozoa present. We have incorporated some unique body fluid markers not used by other groups. We have focused on co-extracting both RNA and DNA that allow for the identification of the body fluid(s) while concurrently obtaining the DNA for profiling from the same sample. In addition, we have tested dried stains with the current screening methods used for the presumptive testing of body fluids to establish if any of these techniques are detrimental to the RNA. We have also looked at aged stains, environmental factors, and the laundering of clothes to investigate the stability of the RNA. We are now validating this multiplex for casework, with a focus on using the established automated DNA-extraction techniques that are currently in use for casework to extract the RNA and DNA. Furthermore, we are concentrating on statistical interpretation of the multiplex when mixed body fluids are present. In particular, we are concentrating on identifying which body fluid the DNA profile originated from when more than one individual DNA profile is obtained from a mixed body fluid stain. doi:10.1016/j.scijus.2009.11.073

Assigning weight of DNA evidence using a continuous model that takes into account stutter and dropout R. Puch-Solisa, T. Claytonb, J. Whitakerb, S. Popec a Forensic Science Service, Birmingham Laboratory, Birmingham Business Park, United Kingdom b Forensic Science Service, Wetherby Laboratory, Wetherby, United Kingdom c Forensic Science Service, London Laboratory, London, United Kingdom Since its inception in the mid-1990s, the application of STR DNA profiling technology to forensic human identification has developed rapidly. Its discriminating power has been extended with the development of larger multiplexes whilst the sensitivity of the method has been increased to a level where a profile can be obtained from almost any biological evidence type. The increased sensitivity has been achieved incrementally by improving DNA-extraction and purification methods, optimisation of the DNA amplification reaction, and via technical

Abstract

improvements to the separation platform and detection instrumentation. If the starting DNA amount is lowered, a number of interpretational challenges follow. It is on these that this presentation will focus. DNA profiles produced from crime samples are affected by a number of wellrecognised and characterised artefacts resulting from the amplification and detection systems used. One phenomenon is stutter. This is caused by slippage of the TAQ polymerase during PCR. Typically this is evident as a small peak one-repeat unit below the larger ‘parent’ peak. Although generally small, it is known that the proportion of the stutter peak relative to the parent peak increases as the amount of starting DNA decreases. Ultimately, this causes interpretational challenges as it may be unclear whether a peak in a stutter position is an artefact or is truly an allele. Furthermore, during amplification of a heterozygous locus, the sister alleles amplify asymmetrically (often referred to as heterozygote imbalance). As the starting DNA amount is progressively reduced, heterozygote imbalance becomes increasingly variable due to stochastic effects leading ultimately to the failure to detect one of the pair of alleles (‘allelic dropout’) or often both alleles (‘locus dropout’). Dropout poses a significant challenge in assigning weight to DNA evidence. Specifically, how does one assess the value of a purported match if a locus in a crime sample lacks one allele seen in the corresponding locus in a reference sample? In this talk we present a probabilistic model for assigning weight to DNA evidence where profiles may be affected by stutter and dropout. The model uses continuous probability distributions estimated from real data generated in our laboratory. The probability distributions allow us to model stutter height and peak imbalance. They dispense with the need to ‘edit out a peak’ as being a stutter and can deal with peak imbalance even in those extreme situations where dropout has to be asserted in order to be considered a ‘match’.

doi:10.1016/j.scijus.2009.11.074

Low template DNA analysis: Further developments supporting its use in the criminal justice process M.J. Greenhalgh Cellmark Forensic Services, Abingdon, United Kingdom Low template DNA analysis has become an increasing part of many forensic investigations. Using a variety of techniques, such as increasing the number of PCR amplification cycles or modification to the capillary electrophoresis detection method, profiles have been obtained from samples containing less than 100 pg of DNA. This has enabled a much wider range of sample types to be analysed and has provided valuable information in many high profile cases. However the results are frequently difficult to interpret owing to stochastic effects and the detection of increased numbers of mixed profiles. The high sensitivity of the technique also increases the possibility that extraneous DNA contamination will be detected. A number of criticisms of the technique led to a review headed by Professor Brian Caddy, A Review of the Science of Low Template DNA Analysis which was published in 2008. The presentation will address the criticisms that have been raised about the reliability and accuracy of low template DNA analysis by reference to studies performed at Orchid Cellmark illustrated with examples from casework. A detailed comparison has been conducted between the two main technical approaches and their advantages and disadvantages will be discussed. Specific studies have also been conducted to justify the use of multiple independent PCR reactions as a method to provide confirmation of results (the Consensus method). The presentation will also include examples of a possible alternative approach to the interpretation of complex mixtures that may be encountered.

doi:10.1016/j.scijus.2009.11.075

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Optimisation of quantum dot-encoded microsphere bioconjugates for single nucleotide polymorphism genotyping S. Thiolleta, S. Higsonb, N. Whitea, S. Morgana a Cranfield University, Cranfield Health – Translational Medicine Group, Cranfield, United Kingdom a Cranfield University, Cranfield Health – Biosensors and Diagnostic Group, Cranfield, United Kingdom

The development of optimised screening assays continues to be an active area of research in the field of molecular diagnostics and forensic genetics. Semiconductor nanocrystals or quantum dots (QDs) have been highlighted as new optimum fluorescent labels due to their unique optical properties. A spectral ‘barcode’ is generated by pulling different QDs in synthetic microspheres which are then illuminated with a single excitation light. In contrast to conventional fluorescent microspheres, QD-doped particles have the potential to produce an unprecedented number of fluorescent codes, with higher chemical- and photo-stability, and lower limits of detection. Surface-capping strategies providing particle solubilisation and biofunctionalisation create a flexible technology for high throughput screening assay. Fluorescent microspheres have previously been used in suspension array platforms for single-strand DNA (ssDNA) identification and quantification, and specifically for allelespecific oligonucleotide (ASO) hybridisation assays. The Luminex platform™ (Austin, TX, USA) represents the most advanced commercial system with a 100-bead library using organic fluorophores. This technology is, however, limited since it requires multiple excitation sources and a specialised detection instrument, the Luminex 100™ analyser. The Luminex platform™ incurs high costs with a limited flexibility of the bead library enlargement because of organic dye spectral overlap. Other ASO methods are time-consuming, costly, or not adapted to multiplex analysis. Microspheres encoded with QDs and produced in large scales (i.e., 50 mg or 20–30 millions of particles) have been developed for single nucleotide polymorphisms genotyping (SNPs). Time, cost, and the biochemistry equipments and processes involved for the assay development were not adapted for an efficient high-throughput assay. An initial study to overcome these limitations was undertaken in our laboratory. A new bioconjugation methodology adapted to commercialised nanocrystals encoded into polystyrene microspheres, the QDEMs (Crystalplex, Pittsburgh, PA, USA) was developed. The impact of the conjugation buffer, the concentration, and the structure of oligonucleotides was evaluated to optimise QDEM bioconjugates. Here, we also optimised the specific hybridisation of ssDNA to QDEM probes in a small liquid format and detected with a QD reporter fluorophore using the ‘Design of Experiments’. Flow cytometers, used as detection method, offer many advantages over the Luminex instruments including low running costs, high flexibility, and highthroughput detection capabilities. QDEM probes were optimised for the detection of ssDNA and for ASO assays with an initial application to the genotyping of two SNPs. QDEM probe technology is expected to enhance suspension array genotyping technology.

doi:10.1016/j.scijus.2009.11.076

Observed and expected numbers of (partially) randomly matching profiles in the Dutch DNA database, and in international DNA searches M.J. Sjerpsa, K.P. van der Beekb, A.D. Kloostermanc a Netherlands Forensic Institute, Digital Technology and Biometry, The Hague, Netherlands b Netherlands Forensic Institute, Custodian Dutch DNA-Database, The Hague, Netherlands c Netherlands Forensic Institute, Human Biological Traces, The Hague, Netherlands