Tim Clayton

Analysis and interpretation of mixed forensic stains using DNA STR profiling

The use of multiplex PCR and fluorescent dye technology in the automated detection and analysis of short tandem repeat loci provides not only qualitative information about the profile--i.e. which alleles are present--but can also provide quantitative information on the relative intensities of the bands, and is therefore a measure of the amount of amplified DNA. The availability of this quantitative information allows for the interpretation of mixtures in a detailed way which has not been previously possible with many other human identification systems. In this paper we present a simple approach to the resolution and analysis of mixed STR profiles resulting from the testing of mixed biological stains in forensic casework and highlight factors which can affect it. This approach requires a detailed knowledge--gained through a mixture of experiments and validation studies--of the behaviour of each locus within the multiplex systems described. We summarise the available data from previously published experimental work and validation studies to examine the general principles underlying this approach.

Interpreting simple STR mixtures using allele peak areas

Although existing statistical models can interpret mixtures qualitatively based upon the alleles present, the use of automated sequencers opens the opportunity to take account of quantitative aspects embodied by the peak area. One step in understanding simple mixtures consisting of just two donors is to estimate the mixture ratio. This is relatively easy to do when four-allele mixtures are evident at a given locus. However, if the mixture consists of three or fewer alleles, the process it is not straightforward. We demonstrate that mixture estimates are consistent across all loci in a multiplex system. Once the mixture ratio is known, then the expected peak areas for any given combination of alleles can be estimated using a simple spreadsheet analysis.

A genetic basis for anomalous band patterns encountered during DNA STR profiling.

Since 1995 the Forensic Science Service (FSS) has carried out DNA profiling of reference samples for the UK National DNA Database and in forensic casework using two multiplex STR profiling systems. During this period, profiles with anomalous banding patterns, although comparatively rare, have been encountered regularly. The FSS has collected instances of triallelic patterns and aberrant diallelic patterns. A systematic examination of these patterns has provided insight into their underlying genetic cause. The triallelic patterns could be classified into two types based on the relative intensities of their component alleles. In the Type 1 pattern the alleles were of uneven intensity, whereas in the Type 2 pattern, all three alleles were of even intensity. Evidence is presented that the more frequent Type 1 pattern is the result of somatic mutation at a heterozygous locus, and the Type 2 pattern is the result of a localized chromosomal rearrangement at a heterozygous locus. Directly from the Type 1 pattern, it was possible to deduce the size difference between the progenitor and mutated allele. All mutational changes were found to be multiples of four nucleotides, suggesting the loss or addition of one or more tetrameric repeat units. Aberrant diallelic patterns were identified by analysts due to an unexpectedly large difference in intensity between alleles at a heterozygous locus. While some of these diallelic patterns are likely caused by the same genetic phenomena described above occurring at a homozygous locus, others are demonstrated to be caused by a mutation in the primer binding sequence, leading to a reduction in amplification efficiency of one allele. It is concluded that based on a visual inspection of a profile, it is possible to infer a likely genetic basis directly from the triallelic pattern. By contrast, the aberrant diallelic patterns can be due to any one of a number of possible genetic effects.

National recommendations of the Technical UK DNA working group on mixture interpretation for the NDNAD and for court going purposes.

The Technical UK DNA working group comprises representatives from all of the major suppliers of the UK and Ireland who contribute to the UK national DNA database. The group has the following terms of reference:To act as a peer review body.To agree experimental designs, to provide advice to the custodian to facilitate the development of the NDNAD.To support the CJS by the development of a coordinated UK strategy.To be inclusive, rather than exclusive, with regard to the introduction and use of methods.To define best scientific practice.To define guidelines for analysis and interpretation of evidence.To produce guidance that can be used by the UK Accreditation Services (UKAS).The group falls under the European Network of Forensic Science Institutes (ENFSI) umbrella. We will feed back recommendations to the ENFSI group for further discussion in order to facilitate European Policy. The group recently met in order to consider in detail the ISFG DNA Commission recommendations on the interpretation of mixtures, to place them in the context of the UK jurisdictions.

Evidential evaluation of DNA profiles using a discrete statistical model implemented in the DNA LiRa software

The high sensitivity of the technology for producing profiles means that it has become routine to produce profiles from relatively small quantities of DNA. The profiles obtained from low template DNA (LTDNA) are affected by several phenomena which must be taken into consideration when interpreting and evaluating this evidence. Furthermore, many of the same phenomena affect profiles from higher amounts of DNA (e.g. where complex mixtures has been revealed). In this article we present a statistical model, which forms the basis of software DNA LiRa, and that is able to calculate likelihood ratios where one to four donors are postulated and for any number of replicates. The model can take into account dropin and allelic dropout for different contributors, template degradation and uncertain allele designations. In this statistical model unknown parameters are treated following the Empirical Bayesian paradigm. The performance of LiRa is tested using examples and the outputs are compared with those generated using two other statistical software packages likeLTD and LRmix. The concept of ban efficiency is introduced as a measure for assessing model sensitivity.

Description of artefacts in the PowerPlex Y23® system associated with excessive quantities of background female DNA

Male on female sexual assault cases that involve azoospermic individuals, those where the male has penetrated but failed to ejaculate, those where there has been an extended interval between the sexual assault and sample collection or where there has been only digital penetration are often difficult to investigate by employing traditional autosomal STR testing. Such cases often involve minimal amounts of male DNA either being deposited initially or remaining after the passage of time. These cases are often further complicated by the presence of large amounts of female DNA compared to the relatively small amounts of male DNA on the intimate samples taken. Y-STR kits provide a solution that allows targeting of male DNA in a mixed male/female sample. However, large quantities of excess female DNA have the potential to generate non-specific artefact peaks. Here we characterise a number of previously reported artefacts observed in the PowerPlex(®) Y23 system. We demonstrate that some of these artefacts can impact on profile interpretation and that they are highly dependent on the levels of female DNA present. These artefacts have been characterised to assist practitioners with the interpretation of such samples.