Andreas O. Tillmar

Analysis of linkage and linkage disequilibrium for eight X-STR markers

X-chromosomal short tandem repeats (X-STR) have proven to be informative and useful in complex relationship testing. The main feature of X-STR markers, compared to autosomal forensic markers, is that all loci are located on the same chromosome. Thus, linkage and linkage disequilibrium may occur. The aim of this work was to study population genetic parameters of eight X-STR markers, located in four linkage groups. We present haplotype frequencies, based on 718 Swedish males, for the four linkage groups included in the Argus X-8 kit. Forensic efficiency parameters have been calculated as well as the allelic association between the tested markers for detection of linkage disequilibrium. To study the occurrences of recombination between the loci, both Swedish and Somali families were typed. A mathematical model for the estimation of recombination frequencies is presented and applied on the family samples. Our study showed that the tested markers all have highly informative forensic values and that there is a significant degree of linkage disequilibrium between the STR markers within the four linkage groups. Furthermore, based on the tested families, we also demonstrated that two of the linkage groups are partially linked. A consequence of these findings is that both linkage and linkage disequilibrium should be accounted for when producing likelihood ratios in relationship testing with X-STR markers.

A universal method for species identification of mammals utilizing next generation sequencing for the analysis of DNA mixtures

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method’s robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.

Effect of linkage between vWA and D12S391 in kinship analysis

Ideally for use in forensic analyses, genetic markers on the same chromosome should be more than 50 Mb in physical distance to ensure full recombination and thus independent inheritance. The forensic community has given attention to two STR markers, D12S391 and vWA, that are 6.3 megabases (Mb) apart on chromosome 12. Recent studies have shown no significant linkage disequilibrium between vWA and D12S391 in U.S. and worldwide populations, although genetic linkage has been identified. It is important to evaluate the impact of linkage effects on kinship analysis. In this study, we aimed to determine a more precise measurement of the recombination frequency between vWA and D12S391 based on a larger number of informative meiosis than has been studied previously. We estimated the recombination frequency (θ) to 0.089 (95% CI 0.044-0.158). Using pedigrees simulated under specific kinship scenarios where recombination was expected to affect the likelihood ratio (LR), we evaluated the impact on LR values of including or ignoring linkage between vWA and D12S391. For all pedigree scenarios considered, on average, LR values ignoring linkage were slightly underestimated than when linkage was considered. However, in the incest scenario considered, LR values could be overestimated up to 25-30 times when linkage was ignored. We demonstrate that the effect of ignoring linkage in the likelihood ratio calculation can be considerable. These results suggest that linkage should be considered during kinship analysis when vWA and D12S391 are tested for pedigrees where a recombination could impact the LR value.

Using X-chromosomal markers in relationship testing: Calculation of likelihood ratios taking both linkage and linkage disequilibrium into account

X-chromosomal markers in forensic genetics have become more widely used during recent years, particularly for relationship testing. Linkage and linkage disequilibrium (LD) must typically be accounted for when using close X-chromosomal markers. Thus, when producing the weight-of-evidence, given by a DNA-analysis with markers that are linked, the normally used product rule is invalid. Here we present an implementation of an efficient model for calculating likelihood ratios (LRs) with markers on the X-chromosome which are linked and in LD. Furthermore, the model was applied on several cases based on data from the eight X-chromosomal loci included in the Mentype(®) Argus X-8 (Biotype). Using a simulation approach we showed that the use of X-chromosome data can offer valuable information for choosing between the alternatives in each of the cases we studied, and that the LR can be high in several cases. We demonstrated that when linkage and LD were disregarded, as opposed to taken into account, the difference in calculated LRs could be considerable. When these differences were large, the estimated haplotype frequencies often had a strong impact and we present a method to estimate haplotype frequencies. Our conclusion is that linkage and LD should be accounted for when using the tested set of markers, and the used model is an efficient way of doing so.

Homogeneity in mitochondrial DNA control region sequences in Swedish subpopulations

In order to promote mitochondrial DNA (mtDNA) testing in Sweden we have typed 296 Swedish males, which will serve as a Swedish mtDNA frequency database. The tested males were taken from seven geographically different regions representing the contemporary Swedish population. The complete mtDNA control region was typed and the Swedish population was shown to have high haplotype diversity with a random match probability of 0.5%. Almost 47% of the tested samples belonged to haplogroup H and further haplogroup comparison with worldwide populations clustered the Swedish mtDNA data together with other European populations. AMOVA analysis of the seven Swedish subregions displayed no significant maternal substructure in Sweden (FST = 0.002). Our conclusion from this study is that the typed Swedish individuals serve as good representatives for a Swedish forensic mtDNA database. Some caution should, however, be taken for individuals from the northernmost part of Sweden (provinces of Norrbotten and Lapland) due to specific demographic conditions. Furthermore, our analysis of a small sample set of a Swedish Saami population confirmed earlier findings that the Swedish Saami population is an outlier among European populations.

FamLink - A user friendly software for linkage calculations in family genetics

The present number of STR loci adopted in relationship testing is chiefly limited to unlinked markers, in most cases residing on different chromosomes. In order to solve more complex cases of relatedness, e.g. deficient paternities and disputed sibships, the number of core loci can be extended. The inclusion of multiple loci on the same chromosome will, however, increase the risk of possible linkage between markers. We present a new software, FamLink, freely available from http://www.FamLink.se, that can perform statistical calculations based on pedigree structures and account for linkage between pairs of markers. In addition, FamLink can simulate genotype data in order to study the effect of accounting for linkage or not. We demonstrate the importance of taking linkage properly into account using examples and real cases.

FamLinkX – implementation of a general model for likelihood computations for X-chromosomal marker data

The use of genetic markers located on the X chromosome has seen a significant increase in the last years and their utility has been well studied. This paper describes the software FamLinkX, freely available at http://www.famlink.se, implementing a new algorithm for likelihood computations accounting for linkage, linkage disequilibrium and mutations. It is obvious that such software is sought for among forensic users as more and more X-chromosomal markers become available. We provide some simulated examples demonstrating the utility of the implementation as well as its application in forensic casework. Though algebraic derivations are generally unfeasible, the paper outlines some theoretical considerations and provides a discussion on the validation of the software. The focus of this paper is to compare the software to existing methods in a forensic setting, perform a validation study as well as to provide an idea of the discriminatory power for X-chromosomal markers.

DNA microarray as a tool in establishing genetic relatedness—Current status and future prospects

In the past decades, microarray technology has definitely put an edge to the field of genetic research. Our aim was to determine whether single nucleotide polymorphism (SNP) microarrays could be used as a tool in establishing genetic relationships where current molecular genetic methods are not sufficient. We used the Genechip, Affymetrix GenomeWide SNP Array 6.0, which detects more than 900,000 SNP markers dispersed throughout the human genome. The intention was to find a good selection of SNP markers that could be used for statistical evaluation of relatedness in a forensic setting. We conducted pairwise comparisons in the R-package FEST as well as pedigree comparisons in Merlin. Our methods were applied on two separate families, where relationships as distant as 3rd cousins were known. In addition, a question about a possible common ancestry between the two families was tested. Relationships as distant as 2nd cousins could be readily distinguished both from unrelated and other, genetically, closer relationships. This was achieved with a selection of 5774 markers, where each pair of markers was separated by a genetic distance of at least 0.5cM (centiMorgan). When considering 3rd cousins, and more distant relationships, the number of markers needs to be extended, consequently decreasing the genetic distance between the markers. However, inclusion of a too large number of markers presents new challenges and our results imply that the use of too dense sets of markers always yields the highest probability for the genetically closest relationship hypothesis. Simulations confirm that this is most probably caused by the fact that the computational model assumes linkage equilibrium between markers, a problem that will be further evaluated. Our results do however suggest that SNP-data derived from microarrays are well suited for kinship determination provided linkage disequilibrium is properly accounted for.

Genetic variation of 15 autosomal STR loci in a Somali population

Allele frequencies for 15 autosomal STR loci included in the AmpFlSTR Identifiler kit (CSF1PO, D13S317, D16S539, D18S51, D19S433, D21S11, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, TH01, TPOX, VWA) were obtained from the analysis of 404 individuals with Somali origin. The overall match probability for the 15 studied loci was 1 in 1.18 x 10(17) and the combined power of exclusion was 0.999997676. Deviation from Hardy-Weinberg equilibrium was observed for locus D2S1338 after correction for multiple testing. When comparing allele frequencies with five other African populations (Karamoja (Uganda), Mozambique, Tanzania and two other Somali population sample sets), only the Somali populations did not show significant differences for any of the tested loci.

DNA Commission of the International Society for Forensic Genetics (ISFG): Guidelines on the use of X-STRs in kinship analysis

Forensic genetic laboratories perform an increasing amount of genetic analyses of the X chromosome, in particular to solve complex cases of kinship analysis. For some biological relationships X-chromosomal markers can be more informative than autosomal markers, and there are a large number of markers, methods and databases that have been described for forensic use. Due to their particular mode of inheritance, and their physical location on a single chromosome, some specific considerations are required when estimating the weight of evidence for X-chromosomal marker DNA data. The DNA Commission of the International Society for Forensic Genetics (ISFG) hereby presents guidelines and recommendations for the use of X-chromosomal markers in kinship analysis with a special focus on the biostatistical evaluation. Linkage and linkage disequilibrium (association of alleles) are of special importance for such evaluations and these concepts and the implications for likelihood calculations are described in more detail. Furthermore it is important to use appropriate computer software that accounts for linkage and linkage disequilibrium among loci, as well as for mutations. Even though some software exist, there is still a need for further improvement of dedicated software.