Forensic Genetic Laboratories Research Articles

First experiences with the Spectrum Compact CE System

The newly released Spectrum Compact CE System by Promega is a capillary electrophoresis instrument developed for DNA-fragment separation and sequencing. In this study, its compatibility to 8 commercial short tandem repeat (STR) kits from 4 different manufacturers, reproducibility (sizing precision, accuracy and concordance) and robustness (sensitivity and mixture resolution) were tested and compared to the ABI PRISM® 310 Genetic Analyzer. The instrument was able to successfully analyse amplicons of all tested kits, proved to be as precise as claimed by manufacturer specifications and was shown to be more robust than the ABI PRISM® 310 Genetic Analyzer in some aspects. Analyses on the Spectrum Compact CE System were able to resolve peaks with length differences of 1-basepair in the short and long fragment range and mixtures of mixture ratios down to 1:30. We describe the advantages and limitations we have observed so far working with this instrument in our forensic genetics laboratory.

A Swiss collaborative exercise for Disaster Victim Identification (DVI): Covering situations with different levels of complexity

The identification of victims of a disaster (DVI) requires the collaboration of different specialists. Within a DVI context, DNA analyses often play an important role. Consequently, forensic genetic laboratories should be prepared to cope with DVI situations, as this can involve large-scale DNA profile comparisons. Six forensic genetic laboratories from Switzerland participated in an exercise where supposedly a plane had crashed. The goal of the exercise was to monitor participants use of dedicated software with ground truth cases and to make them aware of the existence of particular situations that may occur in real cases. For assigning the value of the comparison of the DNA profiles, all participating laboratories used the DVI module of Familias v3.2.1 In addition, one of the 6 laboratories used the Pedigree Searcher from CODIS v7.0. The data (AmpFlSTR® NGM SElect™ profiles) were generated to challenge the participating laboratories: cases with first, second degree biological parents, mutation events, as well as non-paternity cases were included. This study shows that the majority of the participants used the software in an appropriate way. However, a few misleading conclusions were detected for the most challenging situations. These errors belonged to one of the following categories: false pedigree, false association using the higher LR, misleading contextual information (false paternity) and not clustering family members. Specific recommendations are provided in order to reduce misuse of the software and the risk of misinterpretations by using all the relevant information.

A DNA-based method for distinction of fly artifacts from human bloodstains

Fly artifacts resulting from insect activity could act as confounding factors on a crime scene and interfere with bloodstain pattern analysis interpretation. Several techniques have been proposed to distinguish fly artifacts from human bloodstains based on morphological approach and immunological assay, but a DNA-based method has not been developed so far. Even if in forensic genetic investigations the detection of human DNA is generally the primary goal, fly artifacts can provide useful information on the dynamics of crime events. The present study provides a molecular method to detect fly DNA from artifacts deposited by Calliphora vomitoria after feeding on human blood through the analysis of the mitochondrial cytochrome oxidase gene subunit I (COI). Fly artifacts originated from digestive process and of different morphology spanning from red and brownish/light brown, circular and elliptical stains to artifacts with sperm-like tail or a tear-shaped body were collected. The COI amplification was successfully obtained in 94% of fly artifact samples. The method showed high sensitivity and reproducibility, and no human DNA contamination was observed, offering specificity for use in confirmatory test. This molecular approach permits the distinction of fly artifacts from genuine bloodstains and the identification of fly’s species through the COI region sequencing by protocols usually applied in forensic genetic laboratories.

Second GHEP-ISFG exercise for DVI: “DNA-led” victims’ identification in a simulated air crash

The Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) has organized a second collaborative exercise on a simulated case of Disaster Victim Identification (DVI), with the participation of eighteen laboratories. The exercise focused on the analysis of a simulated plane crash case of medium-size resulting in 66 victims with varying degrees of fragmentation of the bodies (with commingled remains). As an additional difficulty, this second exercise included 21 related victims belonging to 6 families among the 66 missings to be identified. A total number of 228 post-mortem samples were represented with aSTR and mtDNA profiles, with a proportion of partial aSTR profiles simulating charred remains. To perform the exercise, participants were provided with aSTR and mtDNA data of 51 reference pedigrees —some of which deficient—including 128 donors for identification purposes. The exercise consisted firstly in the comparison of the post-mortem genetic profiles in order to re-associate fragmented remains to the same individual and secondly in the identification of the re-associated remains by comparing aSTR and mtDNA profiles with reference pedigrees using pre-established thresholds to report a positive identification. Regarding the results of the post-mortem samples re-associations, only a small number of discrepancies among participants were detected, all of which were from just a few labs. However, in the identification process by kinship analysis with family references, there were more discrepancies in comparison to the correct results. The identification results of single victims yielded fewer problems than the identification of multiple related victims within the same family groups. Several reasons for the discrepant results were detected: a) the identity/non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, b) some laboratories failed to use all family references to report the DNA match, c) In families with several related victims, some laboratories firstly identified some victims and then unnecessarily used their genetic information to identify the remaining victims within the family, d) some laboratories did not correctly use “prior odds” values for the Bayesian treatment of the episode for both post-mortem/post-mortem re-associations as well as the ante-mortem/post-mortem comparisons to evaluate the probability of identity. For some of the above reasons, certain laboratories failed to identify some victims. This simulated “DNA-led” identification exercise may help forensic genetic laboratories to gain experience and expertize for DVI or MPI in using genetic data and comparing their own results with the ones in this collaborative exercise.

Comparison between automated DNA extraction employing the EZ1 platform and manual methods using real forensic samples

In the last 30 years, advances in Molecular Biology techniques have allowed its application as an essential tool in the criminal investigation, with a particularly relevant application in the determination of authorship of crimes and the identification of missing persons and unknown corpses. However, with the popularization of Forensic Genetics and the growth of violence, there has been a significant increase in the demand for these tests, which has led Forensic Genetics Laboratories to search for more efficient and safe methods of processing this increasing volume of samples. In this sense, automation was one of the adopted solutions, allowing the processing of the samples more accurately and with the minimum of human interference, reducing the risks, and being a more economical alternative when used in large scale. This paper aims to present the results obtained concerning the standardization of the use of an automated DNA extraction platform and to evaluate the advantages of this method in relation to the manual methods in the extraction of forensic samples from the Laboratory of Forensic Genetics of POLITEC-AP. For the realization of the experiments, 500 samples of DNA were extracted using manual methods (Organic and Chelex 100®) and using the automated extraction equipment EZ1 Advanced XL® from Qiagen ©. The automated extraction using the EZ1® following the manufacturer's protocol or after the adaptation of the protocol presented qualitative and quantitative results superior to those obtained using manual methods, both for reference saliva samples and unknown samples of unknown cadavers (teeth and bones) and secretions collected from victims of sexual violence.

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories.

For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

Validation of a 52-mtSNP minisequencing panel for haplogroup classification of forensic DNA samples.

Mitochondrial DNA (mtDNA) is a useful tool in forensic investigation as it provides information about the matrilineal ancestry of individuals. In addition, mtDNA can be analyzed when the analysis of other nuclear markers is underperforming. Recently, we developed a minisequencing panel for the simultaneous analysis of 52 mtDNA SNPs to classify maternal lineages into the main haplogroups and their phylogeographic origin. In order to make this panel suitable for forensic genetics laboratories, a validation study has been performed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, including species specificity, reproducibility, sensitivity, and stability tests. The results demonstrate that the panel of 52 mtDNA SNPs is highly sensitive, since it enables to obtain complete genetic profiles of samples containing minimal amounts of DNA (1pg). Furthermore, it provides sufficient genetic information to detect the matrilineal biogeographical origin of highly degraded samples, i.e., ancient dating skeletal remains, and samples with the presence of inhibitors, such as hematin and humic acid. In addition, this panel can detect mixtures in samples whose mtDNA haplogroups of contributors are different. Overall, the results of this study demonstrate the suitability of this minisequencing panel of 52 mtDNA SNPs to be used in forensic cases, with samples of low amount or degraded DNA.

Is Genomic DNA extracted and stored at -20°C for long time useful in forensic field?

With the progress of technologies, forensic genetic laboratories have been more frequently involved in "cold case". The aim of this study was to understand whether the DNA extracted and stored for a long time at−20 °C could be useful for new analyses in order to identify the perpetrator of unsolved crimes, especially when evidence is no longer available. We selected 120 DNA samples obtained from evidence collected at crime scene between 2001 and 2010: they were quantified again using Realtime PCR and the “Plexor HY System” kit. The results were compared with those obtained in the past. For 37 samples discrepancies were observed (i.e. positive quantification, identification of male material mixed with female ones). Then these samples were amplified with “AmpFISTR Identifiler Plus” and “PowerPlex ESI 17 Fast System” kits. Genetic profiles useful for a comparison were obtained for all the 37 analysed samples. In 5 samples mixed profiles were highlighted, unlike what was obtained in the past, and in addition, in 28 cases, where no genetic profile was previously typed, a genetic profile was also identified. The study shows how it is possible, even after a considerable time interval, to still obtain genetic profiles useful for a comparison, as well as the possibility of typing new ones. Comparing the results of the analysis of DNA polymorphisms it is possible to underline the improvement that the current techniques have brought to the typing of genetic profiles: regarding the attribution of gender and the perception of contributors within mixtures.

STR analysis of skeletal remains exhumed from a dry “water well” used as a massive clandestine burial in Argentina

The military dictatorship that took power in Argentina from 1976 to 1983, brought enforced disappearance and death to thousands of people in the country. “Pozo de Vargas” is a 3.5 m wide and 60 m deep dry water well used as a massive clandestine burial site in Tucumán, Argentina. The site contained commingled remains as well as partially articulated body parts. Since 2011, EAAF Forensic Genetics Laboratory (LGF-EAAF) has been processing post mortem (PM) samples exhumed from the well to achieve the intra-skeletal re-association and the identification of the bodies or body parts. 981 skeletal remains, with different states of preservation, were analyzed for STR typing using commercial available kits. Different bone elements were exhumed for intra-skeletal re-associations (PM-PM genetic comparisons): teeth, femur and tibia represented more than 65% of sampling and almost all of them were successfully re-associated; spongy bones as scapula, vertebrae and clavicles yielded worse results, although almost 80% of them were also re-associated. 871 skeletal remains (89%) were successfully typed for autosomal STR markers and re-associated into 147 unique STR profiles meaning that at least 147 different individuals were thrown into the water well. After re-association, unique STR profiles obtained from the skeletal remains were compared to ante mortem (AM) family reference database in order to identify victims (AM-PM genetic comparisons), being possible to restore 113 victims to their families. Another challenge of this identification project was the presence of biologically related victims inside the well.

Comparison of DNA yield after long-term storage of Second World War bone samples

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.

The impact of forensic genetics on the management of sexual assault victims: A multicentre GE.F.I project

We conducted a retrospective study to analyse the ways in which sexual abuse was handled by the Italian forensic geneticists in order to delineate common strategies in the management of the collected biological samples.In particular, the results of laboratory analyses were compared with the patients’ reports and the outcome of their medical examination, as recorded on standard evidence collection documents. The purpose of this study is identifying which factors could influence the congruence between what was reported and the typing of male genetic profiles.The project was proposed at the Italian Society of Forensic Genetics and 10 laboratories have joined the study filling in a questionnaire. We analysed 102 cases that occurred between 2006 and 2017. The analysis of the data here presented highlights that the ability to ascertain the presence of male biological material in the collected evidence is not a technical problem for forensic genetics laboratories but it seems to be influenced by other factors, such as how much time elapsed before being taken into care, actions or other relevant events that have occurred between the assault and the medical examination, and the characterisation of the evidence.

Problematic reporting in DNA cases: the need for accredited formats and certified reporting competence

Quality assurance is a pre-requisite for operational forensic genetic laboratories and professional organisations such as ENFSI, SWGDAM or ISFG. These organisations provide guidelines/recommendations for the methods used (both for analysis and evaluation), and for reporting. Aspects regarding analyses will generally be found in the accreditation scope, contrary to evaluation. Moreover, proficiency testing on how to evaluate and report results is still in its infancy. This is problematic as the quality of forensic science services for the administration of justice crucially depends on how results are evaluated and conveyed.In this paper, we use examples of written and oral statements to illustrate how accreditation could improve and ensure that guidelines on interpretation are properly implemented, reviewed and maintained. We underline the need for certifying and validating the knowledge of the experts in that specific field. Indeed, analytical techniques have for many years been the focus of accreditation, rather than the soundness and logic of communication of the value of the results obtained with these techniques. It is now time for a shift and to provide means that ensure that forensic scientists’ conclusions are as justifiable as their analytical methods.

Forensic genetics in sexual assault: A retrospective study on the collection of evidence at the emergency department

The objective of this retrospective study was to examine the accordance between information derived from the written medical report and the results of DNA forensic analyses regarding 122 cases of alleged sexual assault seen at the Emergency Department of Padua Hospital over a period of 5 years. The examination of discrepant results has proved useful to support a broader application of sexual assault management, particularly during the taking of case history. A story of sexual assault with vaginal penetration and ejaculation coincided with the discovery of male DNA using laboratory methods in more than one in every two cases (55%). Medical reports and laboratory findings were also consistent in the four cases in which the patient reported no penetration; no male DNA was found (3% of the whole sample). This led to a total of 71 cases (58% of the whole sample) in which there was a correlation between the victim’s description of the event and the forensic genetics laboratory findings. This study shows a considerable discrepancy between laboratory findings and details recorded in medical reports written at the emergency department in the case of victims of sexual assault and our findings suggest that hospital services which deal with victims of sexual assault should pay more attention to the methods used to obtain and record the material collected during the victim’s medical examination, and to interview patients about the episode. In order to understand the dynamics of the episode in rape cases, and to avoid discrepancies between medical reports and legal reconstructions of sexual crimes, it is crucial to provide victims with support, and to maximize their confidence in the healthcare providers who first attend to them.

Lessons from a study of DNA contaminations from police services and forensic laboratories in Switzerland

In Switzerland, the DNA profiles of police officers collecting crime scene traces as well as forensic genetic laboratories employees are stored in the staff index of the national DNA database to detect potential contaminations. Our study aimed at making a national inventory of contaminations to better understand their origin and to make recommendations in order to decrease their occurrence. For this purpose, a retrospective questionnaire was sent to both police services and forensic genetic laboratories for each case where there was a contamination.Between 2011 and 2015, a total of 709 contaminations were detected. This represents a mean of 11.5 (9.6–13.4) contaminations per year per 1′000 profiles sent to the Swiss DNA database. Feedbacks were obtained from the police, the laboratory or both for 552/709 (78%) of the contaminations. Approximately 86% of these contaminations originated from police officers whereas only 11% were from genetic laboratories employees and 3% were associated to other sources (e.g. positive controls, stain–stain contaminations). Interestingly, a direct contact between the stain and the contaminant person occurred in only 51% of the laboratory contaminations whereas this number increased to 91% for police collaborators. The high level of indirect DNA transfer in laboratories might be explained by the presence of “DNA reservoirs” suggesting that cleaning procedures should be improved. At the police level, most contaminations originated from the person who collected the trace and likely occurred directly at the crime scene. Improving sampling practices could be beneficial to reduce these contaminations.

Working towards implementation of whole genome mitochondrial DNA sequencing into routine casework

Massively parallel sequencing (MPS) technologies offer opportunities to expand forensic mitochondrial DNA (mtDNA) typing capabilities affording higher resolution, an increase in discrimination power, and potentially enabling mixture interpretation. Additionally, improvements to MPS technologies and workflows have made implementation into forensic genetic laboratories increasingly more feasible. Following initial successful studies with the Precision ID mtDNA Whole Genome Panel, the process of internal validation and implementation of whole genome mtDNA sequencing began in our Missing Persons and Forensic Casework Units.

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin.

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories.

Evaluation and comparative analysis on reduction of Globalfiler™ reaction volume in low template samples

In forensic genetic laboratories, low template DNA (LTDNA) samples can be a challenge in the attempting to obtain genetic profiles with the most information possible. In this study, volume reduction on amplification reaction was tested in the kit Globalfiler™, with the intend to optimize the results in LTDNA samples.To carry out this study, several samples extracted with PrepFiler® were diluted successively to very low concentration values. All samples and respective dilutions were amplified with Globalfiler™, for both reaction volumes of 12,5μL and 25μL. Capillary electrophoresis was performed on the ABI PRISM® 3500 Genetic Analyzer and the HID files generated were analysed using GeneMapper® ID-X v.1.4. Forensic DNA samples with very low values of DNA concentration were also tested in the same conditions.The results indicate that the reduction of volume in amplification reaction with Globalfiler™ can often produce higher quality profiles, resulting as an alternative to enhance allelic recovery.

Comparison of manual and automated AmpliSeq™ workflows in the typing of a Somali population with the Precision ID Identity Panel

The Precision ID Identity Panel was used to type 109 Somali individuals in order to obtain allele frequencies for the Somali population. These frequencies were used to establish a Somali HID-SNP database, which will be used for the biostatistic calculations in family and immigration cases. Genotypes obtained with the Precision ID Identity Panel were found to be almost in complete concordance with genotypes obtained with the SNPforID PCR-SBE-CE assay. In seven SNP loci, silent alleles were identified, of which most were previously described in the literature.The project also set out to compare different AmpliSeq™ workflows to investigate the possibility of using automated library building in forensic genetic case work. In order to do so, the SNP typing of the Somalis was performed using three different workflows: 1) manual library building and sequencing on the Ion PGM™, 2) automated library building using the Biomek®3000 and sequencing on the Ion PGM™, and 3) automated library building using the Ion Chef™ and sequencing on the Ion S5™. AmpliSeq™ workflows were compared based on coverage, locus balance, noise, and heterozygote balance. Overall, the Ion Chef™/Ion S5™ workflow was found to give the best results and required least hands-on time in the laboratory. However, the Ion Chef™/Ion S5™ workflow was also the most expensive. The number of libraries that may be constructed in one Ion Chef™ library building run was limited to eight, which is too little for high throughput workflows. The Biomek®3000/Ion PGM™ workflow was found to perform similarly to the manual/Ion PGM™ workflow. This argues for the use of automated library building in forensic genetic case work. Automated library building decreases the workload of the laboratory staff, decreases the risk of pipetting errors, and simplifies the daily workflow in forensic genetic laboratories.

LIMS configuration to fit new massively parallel sequencing workflows in forensic genetics

Laboratory Information Management Systems (LIMS) are valuable tools in the forensic genetics laboratory, streamlining sample and analysis traceability, batching of multiple samples, integration and data transfer with genetic analyzers, monitoring of test results, DNA data exchange with National DNA databases, and flexible reporting. This work, developed in the framework of the DNASEQEX project, presents the configuration of a commercial LIMS system to fit the automated workflow provided by the IonChef/Ion S5XL platforms and the Torrent Server Suite/Converge software packages for the massively parallel sequence analysis of STR markers in the forensic lab. LIMS configurations included (1) the setup of analysis and batch templates for DNA library preparation, and DNA library quantification (2) the creation of composite samples for MPS-STR library tracking, (3) the development of files to electronically export MPS sample setups and analysis to the Torrent Server Software, (4) the configuration of scripts for the electronic transfer into the LIMS system of STR allele and sequence data files generated by the Converge software, and (5)the development of DNA data export files to national DNA databases. The developed LIMS configuration provides the laboratory with the tools required for setting up automated MPS workflows, helping to efficiently and accurately acquire, manage, and exchange MPS data in the forensic genetics laboratory.

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.