Jo-Anne Bright

The interpretation of single source and mixed DNA profiles

A method for interpreting autosomal mixed DNA profiles based on continuous modelling of peak heights is described. MCMC is applied with a model for allelic and stutter heights to produce a probability for the data given a specified genotype combination. The theory extends to handle any number of contributors and replicates, although practical implementation limits analyses to four contributors. The probability of the peak data given a genotype combination has proven to be a highly intuitive probability that may be assessed subjectively by experienced caseworkers. Whilst caseworkers will not assess the probabilities per se, they can broadly judge genotypes that fit the observed data well, and those that fit relatively less well. These probabilities are used when calculating a subsequent likelihood ratio. The method has been trialled on a number of mixed DNA profiles constructed from known contributors. The results have been assessed against a binary approach and also compared with the subjective judgement of an analyst.

Characterising stutter in forensic STR multiplexes

Stutter is an artefact seen when amplifying short tandem repeats and typically occurs at one repeat unit shorter in length than the parent allele. In forensic analysis, stutter complicates the analysis of DNA profiles from multiple contributors, known as mixed profiles, a common profile type. Consequently it is important to both understand and predict stutter behaviour in order to improve our understanding of the resolution and interpretation of these profiles. Whilst stutter is well recognised and documented, little information is available that identifies and quantifies what influences the formation of stutter. In this work we use a novel approach to examine this. We have used synthetic oligonucleotides comprising multiple repeat units to test; the influence of repeat number, the influence of repeat sequence and the impact of interruptions to the repeat sequence length. Using multiple replicates allows detailed statistical analysis. We have confirmed a linear relationship between stutter ratio and repeat number. We have shown that increased A-T content increases stutter ratio and that interruptions in repeating sequences decreased stutter ratios to levels similar to the longest uninterrupted repeat stretch. We also found that there was no relationship between stutter ratio and repeat number for a repeat unit with an A-T content of 1/4 and that half of the interrupted repeat sequences stuttered significantly less than their longest uninterrupted repeat stretches. We have applied the knowledge gained to examine specific features of the loci present in the AmpFlSTR(®) SGM Plus(®) multiplex kit used in our laboratory.

Developing allelic and stutter peak height models for a continuous method of DNA interpretation

Traditional forensic DNA interpretation methods are restricted as they are unable to deal completely with complex low level or mixed DNA profiles. This type of data has become more prevalent as DNA typing technologies become more sensitive. In addition they do not make full use of the information available in peak heights. Existing methods of interpretation are often described as binary which describes the fact that the probability of the evidence is assigned as 0 or 1 (hence binary) (see for example [1] at 7.3.3). These methods are being replaced by more advanced interpretation methods such as continuous models. In this paper we describe a series of models that can be used to calculate expected values for allele and stutter peak heights, and their ratio SR. This model could inform methods which implement a continuous method for the interpretation of DNA profiling data.

Examination of the variability in mixed DNA profile parameters for the Identifiler™ multiplex

The interpretation of mixed DNA profiles is often undertaken by determining to what extent possible genotype combinations may be eliminated from consideration. This requires an understanding of the variability in height or area for the two alleles of a heterozygote (heterozygote balance), and in the variability of mixture proportion or mixture ratio across loci. We present here empirical data to help assess the magnitude of this variability for Identifiler profiles. We find that heterozygote balance is affected by peak height as expected and that at heights above 267RFU the two peaks of a heterozygote demonstrates a peak height ratio between 0.6 and 1.66. For mixtures we introduce a concept, APH, the average peak heights of the active alleles. Above an APH of 110RFU mixture proportion demonstrated variation no more than 0.2 from the average across loci. Mixture ratio variability was affected by both the mixture ratio, M(r) per se and by the APH. The function (2APH/(1+M(r))) appears to be a reasonable predictor of the variability and approximates the expected peak height of the minor alleles. At (2APH/(1+M(r)))> or =300RFU the mixture ratio at a locus is expected to be within a factor of 2 of the average across loci.

An evaluation of potential allelic association between the STRs vWA and D12S391: Implications in criminal casework and applications to short pedigrees

An evaluation was carried out to determine the effect on routine forensic calculations when incorporating STRs D12S391 and vWA. These loci are co-located on the same arm of chromosome 12. It has been suggested that allelic association could result in over-estimates of strength-of-evidence calculations. In the first place, we argue that is very unlikely that genotypes collected from typical cosmopolitan forensic databases can provide meaningful information about effects attributable to physical linkage. Since admixture is the most likely cause of allelic association in modern populations we specifically evaluate this effect. We use computer simulation as the preferred approach to generate populations with disequilibrium and observe the effect on match probability. Although we have specifically evaluated the linkage between D12S391 and vWA, the methods described in this paper can be extended and generalized to evaluate linkage effects between any pair of loci where the recombination rate is known. Many jurisdictions apply a subpopulation correction following the standard method of Balding and Nichols. Such corrections would appear to be more than adequate to compensate for any increase in match probability that we were able to create by this admixture. Linkage is likely to have an appreciable effect on relatedness calculations in short pedigrees in some but not all instances. We examined those circumstances where an effect is likely and give formulae for some common situations. The complexity of these calculations is a cause for concern in some laboratories. We discuss possible strategies that might be employed and plausible effects.

Comparison of the variables affecting the recovery of DNA from common drinking containers

As the boundaries of forensic DNA profiling continue to expand, less obvious sources of biological evidence are being collected at crime scenes for DNA profiling. One example is the recovery of biological evidence from common drink containers, such as bottles and cans, which have been found at crime scenes. There are many variables that may have an impact on recovering a DNA profile from such exhibits. In this research, the effects of person to person variation, time, type of drink (including alcoholic and non-alcoholic beverages), and type of drink container, were assessed for their impact on the major analytical outcomes of the DNA process. The results show that the alpha-amylase activity varies from individual to individual and is reduced in the presence of some alcoholic drinks. A reasonable DNA yield was obtained from all samples, however, the concentrations exhibited significant person to person variation. The type of drink container influenced the DNA yield with cans giving a higher yield than bottles of the same drink type. To a reduced extent the presence or absence of alcohol affected the overall DNA yield and when partial or failed DNA profiles were produced they were more likely to be associated with alcoholic drinks than non-alcoholic drinks.

A comparison of statistical models for the analysis of complex forensic DNA profiles

Complex mixtures and LtDNA profiles are difficult to interpret. As yet there is no consensus within the forensic biology community as to how these profiles should be interpreted. This paper is a review of some of the current interpretation models, highlighting their weaknesses and strengths. It also discusses what a forensic biologist requires in an interpretation model and if this can be realistically executed under current justice systems.

Degradation of forensic DNA profiles

Selected profiles typed at the Promega PowerPlex 21 (PP21) loci were examined to determine if a linear or exponential model best described the relationship between peak height and molecular weight. There were fewer large departures from observed and expected peak heights using the exponential model. The larger differences that were observed were exclusively at the high molecular weight loci. We conclude that the data supports the use of an exponential curve to model peak heights versus molecular weight in PP21 profiles. We believe this observation will improve our ability to model expected peak heights for use in DNA interpretation software.

A comparison of stochastic variation in mixed and unmixed casework and synthetic samples.

Understanding the behaviour of mixed DNA profiles is of paramount importance in forensic DNA analysis. Key parameters are those of heterozygote balance and mixture proportion and its variability. These parameters have been previously explored as a function of the average peak height of the active alleles in single source and mixed samples derived from pristine DNA. Here we report a comparison of this data with data obtained from casework samples. This allows an assessment of the difference in the distribution of heterozygote balance between mixed and single source stains and between casework mixtures and synthetic mixtures constructed from pristine DNA.

The interpretation of low level DNA mixtures.

The occurrence of mixed DNA profiles in forensic samples is not uncommon. Interpretation of these profiles however can be challenging. In this paper we compare three different models for interpreting mixed DNA profiles prior to the calculation of a statistical weight. Two of these models take into account the peak height of the alleles. The third method uses an unconstrained combinatorial approach. We compare the statistical weights calculated after applying the three different models to one low level two-person mixed DNA profile derived from a crime sample and provide tables of equations that can be applied to many different scenarios including single source and two and three person mixed DNA profiles.