Ate D. Kloosterman

PCR-amplification and detection of the human D1S80 VNTR locus Amplification conditions, population genetics and application in forensic analysis

SummaryA series of experiments has been performed to evaluate amplification and typing of the D1S80 VNTR locus. The validation study that has been carried out showed that correct D1S80 typing results can be obtained when a defined amplification protocol and a high-resolution polyacrylamide gel electrophoresis method are used. The use of the Chelex extraction protocol has substantially reduced the processing time. DNA-extraction, amplification and subsequent typing can be performed in one day. The discrimination power of this locus is 0.94 in a Dutch Caucasian population sample. The system is extremely sensitive: 0.1 ng of genomic DNA gave a correct typing result. The test could also detect the correct genotypes in mixed samples containing DNA from different individuals. Even if the major type was in a 20-fold excess, the minority type could still be amplified and typed correctly. We have found no deviation from Hardy-Weinberg equilibrium in a Dutch Caucasian population sample. Evidence for the somatic stability of this locus was obtained from a set of experiments where we compared DNA-profiles from corresponding blood, semen and saliva samples. The results of this study suggest that in the near future analysis of the D1S80 locus by DNA-amplification can be applied in actual forensic case work.ZusammenfassungEine experimentelle Serie wurde durchgeführt, um die Amplifikations- und Typisierungsbedingungen des VNTR-Locus D1S80 festzulegen. Die Validierungsstudie, welche durchgeführt wurde, zeigte, daß korrekte Typisierungsergebnisse erhalten werden können, wenn ein definiertes Amplifikationsprotokoll und eine hochauflösende polyacrylamidgel-elektrophoretische Methode benutzt werden. Der Gebrauch des Chelex-Extraktionsprotokolls hat die Bearbeitungszeit erheblich reduziert. Die DNA-Extraktion, Amplifikation und anschließende Typisierung können an einem Tag durchgeführt werden. Die Diskriminationskraft dieses Locus ist 0,94 in einer holländischen Populationsstichprobe (Europäer). Das System ist extrem empfindlich: 0,1 ng genomischer DNA ergaben ein korrektes Typisierungsergebnis. Der Test war auch geeignet, korrekte Bestimmung der Genotypen in gemischten Samples durchzuführen, welche DNA von verschiedenen Personen enthielten. Auch wenn der Haupttyp in 20-fachem Überschuß vorhanden war, konnte der Nebentyp noch amplifiziert und korrekt bestimmt werden. In einer holländischen Populationsstichprobe haben wir keine Abweichung vom Hardy-Weinberg-Gleichgewicht gefunden. Evidenz für somatische Stabilität dieses Locus wurde dadurch erhalten, daß ein Satz von Experimenten durchgeführt wurde, in welchen die DNA-Profile korrespondierender Blut-, Samen- und Speichelproben verglichen wurden. Die Ergebnisse dieser Untersuchung legen nahe, daß in der nahen Zukunft die Analyse des D1S80-Locus durch DNA-Amplifikation im aktuellen forensischen Fallmaterial durchgeführt werden kann.

Homogeneity and distinctiveness of Polish paternal lineages revealed by Y chromosome microsatellite haplotype analysis

Abstract. Different regional populations from Poland were studied in order to assess the genetic heterogeneity within Poland, investigate the genetic relationships with other European populations and provide a population-specific reference database for anthropological and forensic studies. Nine Y-chromosomal microsatellites were analysed in a total of 919 unrelated males from six regions of Poland and in 1,273 male individuals from nine other European populations. AMOVA revealed that all of the molecular variation in the Polish dataset is due to variation within populations, and no variation was detected among populations of different regions of Poland. However, in the non-Polish European dataset 9.3% (P<0.0001) of the total variation was due to differences among populations. Consequently, differences in RST-values between all possible pairs of Polish populations were not statistically significant, whereas significant differences were observed in nearly all comparisons of Polish and non-Polish European populations. Phylogenetic analyses demonstrated tight clustering of Polish populations separated from non-Polish groups. Population clustering based on Y-STR haplotypes generally correlates well with the geography and history of the region. Thus, our data are consistent with the assumption of homogeneity of present-day paternal lineages within Poland and their distinctiveness from other parts of Europe, at least in respect to their Y-STR haplotypes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00439-002-0728-0.

New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains

In forensic science, the unequivocal identification of the cellular origin of crime scene samples used for DNA profiling can provide crucial information for crime scene reconstruction. We have previously shown that various mRNA markers from genes with expression patterns specific for blood and saliva can be established from whole-genome expression analysis of time-wise degraded samples and were stable enough to specifically identify blood and saliva stains up to 180 days of age. Here, we showed that nine blood-specific and five saliva-specific mRNA markers can be amplified successfully and reliably in much older blood (13–16 years) and saliva (2–6 years) stains, respectively, suggesting their suitability for tissue identification in forensic case work. Moreover, our findings imply that forensic RNA testing can be reliable and robust if degraded samples are considered in the marker ascertainment procedure, with promising expectations beyond tissue identification purposes.

Post-coital vaginal sampling with nylon flocked swabs improves DNA typing

In the examination of sexual assault cases, DNA typing of vaginal samples mostly occurs after differential DNA extraction. Notwithstanding the differential extraction method, the DNA profiles from the seminal fraction often show the male alleles at low-level in combination with female alleles. This unfavorable ratio male to female DNA is due to a limited amount of sperm cells and an overwhelming quantity of female cells. In this study, we compared standard cotton and nylon flocked swabs for post-coital vaginal sampling. Twelve couples donated 88 vaginal swabs - 44 cotton, 44 nylon flocked - which were taken with a time since intercourse (TSI) up to 84 h. These vaginal swabs were sorted into categories on the basis of the TSI and submitted to (1) microscopic examination for the presence of male cells, (2) presumptive tests for the detection of seminal fluid and (3) DNA typing. Cellular elution was found to be 6-fold more efficient from the nylon flocked swabs. This makes microscopic analysis less time consuming as the higher cell yield and better cell morphology simplify detection of male cells. Both swab types reveal similar results regarding presumptive tests and male DNA typing. Positive presumptive tests (RSID-semen and PSA) were obtained up to 60 h TSI and male autosomal profiles up to 72 h TSI. Interestingly, over 50% of the samples negative for both presumptive tests resulted in informative male STR profiles. After differential extraction, less DNA was left on the nylon flocked swabs and more male DNA was isolated. Our results imply that the use of nylon flocked swabs for vaginal sampling will improve microscopic analysis and DNA typing in the medical forensic investigation of sexual assault cases.

Developing a set of ancestry-sensitive DNA markers reflecting continental origins of humans

BackgroundThe identification and use of Ancestry-Sensitive Markers (ASMs), i.e. genetic polymorphisms facilitating the genetic reconstruction of geographical origins of individuals, is far from straightforward.ResultsHere we describe the ascertainment and application of five different sets of 47 single nucleotide polymorphisms (SNPs) allowing the inference of major human groups of different continental origin. For this, we first used 74 cell lines, representing human males from six different geographical areas and screened them with the Affymetrix Mapping 10K assay. In addition to using summary statistics estimating the genetic diversity among multiple groups of individuals defined by geography or language, we also used the program STRUCTURE to detect genetically distinct subgroups. Subsequently, we used a pairwise FST ranking procedure among all pairs of genetic subgroups in order to identify a single best performing set of ASMs. Our initial results were independently confirmed by genotyping this set of ASMs in 22 individuals from Somalia, Afghanistan and Sudan and in 919 samples from the CEPH Human Genome Diversity Panel (HGDP-CEPH)ConclusionBy means of our pairwise population FST ranking approach we identified a set of 47 SNPs that could serve as a panel of ASMs at a continental level.

Error rates in forensic DNA analysis: Definition, numbers, impact and communication

Forensic DNA casework is currently regarded as one of the most important types of forensic evidence, and important decisions in intelligence and justice are based on it. However, errors occasionally occur and may have very serious consequences. In other domains, error rates have been defined and published. The forensic domain is lagging behind concerning this transparency for various reasons. In this paper we provide definitions and observed frequencies for different types of errors at the Human Biological Traces Department of the Netherlands Forensic Institute (NFI) over the years 2008-2012. Furthermore, we assess their actual and potential impact and describe how the NFI deals with the communication of these numbers to the legal justice system. We conclude that the observed relative frequency of quality failures is comparable to studies from clinical laboratories and genetic testing centres. Furthermore, this frequency is constant over the five-year study period. The most common causes of failures related to the laboratory process were contamination and human error. Most human errors could be corrected, whereas gross contamination in crime samples often resulted in irreversible consequences. Hence this type of contamination is identified as the most significant source of error. Of the known contamination incidents, most were detected by the NFI quality control system before the report was issued to the authorities, and thus did not lead to flawed decisions like false convictions. However in a very limited number of cases crucial errors were detected after the report was issued, sometimes with severe consequences. Many of these errors were made in the post-analytical phase. The error rates reported in this paper are useful for quality improvement and benchmarking, and contribute to an open research culture that promotes public trust. However, they are irrelevant in the context of a particular case. Here case-specific probabilities of undetected errors are needed. These should be reported, separately from the match probability, when requested by the court or when there are internal or external indications for error. It should also be made clear that there are various other issues to consider, like DNA transfer. Forensic statistical models, in particular Bayesian networks, may be useful to take the various uncertainties into account and demonstrate their effects on the evidential value of the forensic DNA results.

Development of a mRNA profiling multiplex for the inference of organ tissues

Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.

Population data of the HLA DQ alpha locus in Dutch Caucasians. Comparison with other population studies.

SummaryThe HLA DQa amplification and typing kit has been designed to be used by the forensic community for purposes of identity testing. The introduction of any new DNA marker in forensic identity testing requires the establishment of a population database for the relevant population(s) [1]. To this end allele and genotype frequencies for the HLADQα locus were determined in a Dutch Caucasian population sample and compared with 7 other population genetic studies. In our population sample the HLA DQα genotype frequencies did not deviate from Hardy-Weinberg expectations and for this locus the power of discrimination is 0.94. A test for homogeneity of the HLA DQα population data based on the allele frequency counts for 8 Caucasian population samples was performed and significant differences were found (P = 0.007) . The differences in the frequency of the HLADQα 2 and 3 alleles are the major cause of this deviation. No deviation from population homogeneity was observed when we compared thegenotype frequency distributions among the 8 Caucasian population samples. Combined with the extensive validation studies from Comey and Budowle [7] and Helmuth et al. [8] this population genetic study will allow HLADQα typing to be used in forensic identity testing in the Netherlands.ZusammenfassungDer Kit für die Amplifikation und Typisierung von HLA DQa wurde als geeignet erklärt für Identitätstests. Die Einführung jedes neuen DNA-Markersystems in forensischen Identitätsuntersuchungen erfordert die Etablierung einer Datenbank für die relevante(n) Population(en) [1]. Mit dieser Zielsetzung wurden Allel- und Genotypfrequenzen in einer holländisehen Kaukasier-Stichprobe untersucht und mit 7 anderen populationsgenetischen Studien verglichen. In unserer Populationsstichprobe wichen die HLADQα-Genotypfrequenzen nicht von Hardy-Weinberg-Erwartungen ab; und für diesen Locus beträgt die Diskriminations-Kraft 0.94. Eine Untersuchung auf Homogenität der HLADQα-Populationsdaten, basierend auf den Allel-frequenzen für 8 kaukasische Populationsstichproben, wurde durchgeführt und es fanden sich signifikante Unterschiede (P = 0.007). Die Differenzen in der Frequenz der Allele HLADQα 2 und 3 sind die wesentliche Ursache dieser Abweichung. Keine Abweichung von der Homogenität wurde beobachtet, wenn wir die Genotyp-Frequenzverteilungen zwischen den 8 kaukasischen Populationsstichproben verglichen. In Kombination mit den extensiven Validierungsstudien von Comey und Budowle [7] und Helmuth et al. [8] erlaubt die vorliegende populationsgenetische Studie die Typisierung von HLA DQα bei forensischen Identitätsuntersuchungen in den Niederlanden.The HLA DQa amplification and typing kit has been designed to be used by the forensic community for purposes of identity testing. The introduction of any new DNA marker in forensic identity testing requires the establishment of a population database for the relevant population(s) [1]. To this end allele and genotype frequencies for the HLADQα locus were determined in a Dutch Caucasian population sample and compared with 7 other population genetic studies. In our population sample the HLA DQα genotype frequencies did not deviate from Hardy-Weinberg expectations and for this locus the power of discrimination is 0.94. A test for homogeneity of the HLA DQα population data based on the allele frequency counts for 8 Caucasian population samples was performed and significant differences were found (P = 0.007) . The differences in the frequency of the HLADQα 2 and 3 alleles are the major cause of this deviation. No deviation from population homogeneity was observed when we compared thegenotype frequency distributions among the 8 Caucasian population samples. Combined with the extensive validation studies from Comey and Budowle [7] and Helmuth et al. [8] this population genetic study will allow HLADQα typing to be used in forensic identity testing in the Netherlands.

On the consequences of DNA profile mismatches for close relatives of an excluded suspect

Abstract If the DNA profiles of a crime stain and the reference sample from the suspect do not match, the suspect is excluded as the donor of the crime stain. However, in some situations the DNA evidence can suggest that a close relative of the suspect might match the stain, in particular when the reference sample from the suspect and the crime stain share rare alleles. This finding can be important for the authorities. The forensic scientist has to decide whether or not to notify the authorities in these circumstances. To the best of our knowledge there is not yet an objective rule for making this decision. We propose such a decision rule for brothers of the suspect, investigate its performance and address some ethical, legal, and practical aspects. Our calculations can be simply adjusted for other relatives of the suspect.

A Dutch population study of the STR Loci HUMTHO1, HUMFES/FPS, HUMVWA31/1 and HUMF13A1, conducted for forensic purposes.

We report on a Dutch population study of the STR loci HUMTHOI, HUMFES/FPS, HUMVWA31/1, and HUMF13A1, in which we used multiplex amplification and automated fragment detection. Genotype and allele frequencies showed no deviation from Hardy-Weinberg and linkage equilibrium. The improved Bonferroni procedure was used to combine the results of several tests. The power of discrimination of a complete profile exceeded 0.9998. We compared the allele frequencies in the Dutch sample to the frequencies in other populations using a biplot to visualize alleles and populations simultaneously. The Dutch sample was similar to most other Caucasian samples. The data demonstrate that the genetic systems in this report are a valuable tool for forensic identity testing in The Netherlands.