The muzzle to target distance -staining inside different parts of the firearm barrel.

Biological traces inside firearm barrels were observed as a result of contact shots to the head. The present study was conducted to investigate the influence of the muzzle to target distance on staining inside the anterior and posterior part of firearm barrels. Ninety-nine shots were fired to so-called reference cubes (10% gelatine, 12 cm edge length, embedded paint-blood-pad) using three current handguns. Shot range was varied from contact to 50 cm distance. High-speed cameras recorded external backspatter. Endoscopic examination assessed visible staining along the barrel. Each two swabbings were gathered from the anterior and the posterior part of the barrel. The first swabs were submitted to quantitative PCR, the second ones to DNA-RNA-co-extraction. Thorough mechanical and chemical cleaning was performed to avoid any contamination which was controlled by negative zero swabs after each cleaning. In single shots up to 50 cm distance, minimal, but DNA-positive sporadic traces were detected inside the barrel in vicinity of the muzzle. Visible complex staining varying in extent was observed in the anterior barrel part for 10 cm or less distance in dependence of the calibre. The posterior part showed detectable traces only after close range shots (< 5 cm). Generally staining inside the barrel decreased from the muzzle to the rear end, which correlated with the yield of DNA. Some contact shots did not cause any staining in the posterior part of the barrel despite massive external backspatter. Blood-specific miRNA was primarily found where DNA was detected. This experience encourages to take a second swab for RNA analysis. The amount of nucleic acids in the barrel at varying muzzle to target distances is subject to large variations between individual shots and therefore appears not suitable for a reliable determination of the shot distance in a particular case on its own. Instead, shot range estimation should also take into account morphology and distribution of traces inside the barrel.

Self- and non-self-DNA on hands and sleeve cuffs.

Studying DNA transfer and persistence has become increasingly important over the last decade, due to the impressive sensitivity of modern DNA detection methods in forensic genetics. To improve our understanding of background DNA that could also potentially be transferred, we analyzed the DNA composition on the outside of sleeve cuffs and sampled DNA directly from the hands of four different collaborators upon their arrival at work during 25 working days. Sampling of their hands was repeated after several hours working in our department. The shedder status of the participants, as assumed from previous internal studies, was well re-produced in the study. However, we noticed that the DNA shedding capacity could also change drastically during the day, with one participant showing a more than sixfold increase between hands sampled in the morning and hands sampled in the afternoon. As expected, poor DNA shedders carry more relative amounts of non-self-DNA on their hands than good shedders. Non-self-alleles were detected in 95% of the samples. We also observed potential effects of hand washing and the mode of transport to get to work on the DNA amount. People living with family members occasionally carried their DNA on their hands and more frequently on their sleeve cuffs. Sleeve cuffs, as being close to our hands, have a large potential to transfer DNA from one place to another, yet they have sparsely been studied as DNA transfer intermediates so far. In general, we collected consistently more DNA from the sleeve cuffs than from the hands of the participants, demonstrating their importance as potential transfer vectors. More DNA was recovered from sleeve cuffs made of synthetic fabric than from cuffs made of cotton or leather. In the afternoon, DNA from co-habitant family members could not be detected on the hands anymore and the detection of profiles from colleagues became more frequent. From two out of 100 analyzed sleeve cuffs and two out of 200 sampled hands, we established unknown major DNA profiles that would have been suitable for an entry in the national DNA database. This finding demonstrates the possibility to transfer DNA that has most likely been picked up somewhere in the public space.

"Total Human DNA Sampling" - Forensic DNA profiles from large areas.

Employed for the first time in 1986, DNA profiling is nowadays established as one of the most widely used forensic techniques worldwide. However, until today, no efficient sampling technique existed to collect DNA from human skin cells from a large area, not to say from the floor of an entire room. This has been extremely unfortunate, as there is enormous forensic potential in these DNA traces from the ground to provide clues as to who has been present at a particular location, i.e. at the crime scene. By desquamation, humans loose several millions of skin cells per day, everywhere they stand, sit or walk; and they can do little about it. We developed a fast and simple method by which we can make use of all those lost skin cells. We use a vacuum cleaner equipped with a specialized filter cartridge to sample the ground. Fragmentation of the filter membrane and subsequent parallel processing of the filter fragments, using a modified Chelex® 100 extraction protocol, significantly reduce the complexity of the dust mixture. In this way, a large number of interpretable major contributor DNA profiles can be generated from individuals who have been present on the sampled surface. Overall, at least 38 % of the generated DNA profiles from all sampled test areas fulfilled the criteria for submission of single major contributor profiles to the Swiss DNA database. As demonstrated through a mock crime scene scenario simulating an indoor stabbing event, the perpetrator's DNA could be found on the floor even after a very short stay in the room of less than one minute. Furthermore, already the first application of the method at a real crime scene led to relevant case information for the police. Given its large investigative potential, we recommend Total Human DNA Sampling as a helpful complemental forensic tool to conventional DNA trace collection in major crimes.

A holistic approach for the selection of forensic DNA swabs.

In the present study, we compared the performance of five different ISO 18385 certified forensic swabs for DNA sampling in practice over a time period of five months. Comparisons were made for DNA profiling success rates, measured as the percentage of CODIS (Combined DNA Index System) suitable profiles as well as for practical suitability during sampling at the scene, measured through a survey among collaborators. More than forty members of our crime scene investigation (CSI) unit took part in the test series and provided structured feedback concerning different aspects of swab handling. A total number of 1094 "touch" DNA samples have been subjected to DNA analysis. Swabs performed significantly different in terms of DNA profiling success rates. We also observed significant differences in DNA extraction efficiency between swabs. The evaluation by the collaborators of various aspects of handling differed significantly between swabs. We can assume that a more convenient handling decreases the risk of contamination or sample mislabelling and increases sampling efficiency and staff satisfaction. Our results demonstrate that the selection of disposable sampling devices such as forensic swabs for DNA sampling should be made based on a holistic approach. To be able to select the best performing swab for a given combination of CSI and DNA laboratory procedures, it might not be sufficient to only perform DNA extraction comparisons and trace sampling under controlled laboratory conditions.

Human background DNA on stones in an urban environment.

Stones are frequently used as tools in criminal acts. In our department, around 5 % of all analysed crime scene related trace samples are contact or touch DNA traces swabbed from stones. These samples are primarily related to cases of damage to property and burglary. In court, questions can arise about DNA transfer and the persistence of background DNA not related to the respective crime. To shed some light on the question of how likely it is to detect human DNA as background DNA on stones from an urban environment, the surfaces of 108 stones sampled throughout the city of Bern, the Swiss capital, were swabbed. We detected a median quantity of 33 pg on the sampled stones. STR-profiles suitable for a CODIS (Combined DNA Index System) registration in the Swiss DNA database were established from 6.5 % of all sampled stone surfaces. For comparison, retrospective casework data analysis from routine crime scene samples demonstrates a success rate of 20.6 % for the establishment of CODIS-suitable DNA profiles from stones sampled for touch DNA. We further investigated how climatic conditions, location and properties of the stones affected the quantity and quality of the recovered DNA. In this study, we show that the quantity of the measurable DNA decreases significantly with increasing temperature. Furthermore, less DNA could be recovered from porous stones, compared to smooth ones.

Recommendations for the successful identification of altered human remains using standard and emerging technologies: Results of a systematic approach.

Successful DNA-based identification of altered human remains relies on the condition of the corpses and varies between tissue types. Therefore, the aim of this prospective multicenter study was to generate evidence-based recommendations for the successful identification of altered remains. For this, 19 commonly used soft and hard tissues from 102 altered human bodies were investigated. The corpses' condition was categorized into three anatomical regions using a practical scoring system. Besides other data, DNA yields, degradation indices, and short tandem repeat (STR) profile completeness were determined in 949 tissue samples. Additionally, varying degrees of alteration and tissue-specific differences were evaluated using the Next Generation Sequencing (NGS) platform MiSeq FGx™. Selected challenging samples were sequenced in parallel with the Ion S5™ platform to assess platform-specific performances in the prediction of the deceased's phenotype and the biogeographic ancestry. Differences between tissue types and DNA extraction methods were found, revealing, for example, the lowest degradation for vertebral disc samples from corpses with initiating, advanced and high degrees of decomposition. With respect to STR profile completeness, blood samples outperformed all other tissues including even profoundly degraded corpses. NGS results revealed higher profile completeness compared to standard capillary electrophoresis (CE) genotyping. Per sample, material and degradation degree, a probability for its genotyping success, including the "extended" European Standard Set (eESS) loci, was provided for the forensic community. Based on the observations, recommendations for the alteration-specific optimal tissue types were made to improve the first-attempt identification success of altered human remains for forensic casework.

Forensic DNA phenotyping in Europe: How far may it go?

The fast evolution of genetic sequencing techniques led to new applications in forensic genetics, one of these being the prediction of the physical appearance of a possible perpetrator from biological traces found at the crime scene. Some European countries recently changed their legislations, to permit this technique, also known as Forensic DNA Phenotyping (FDP). The phenotypical traits that may be analyzed under those revised domestic laws are usually restricted to include no information about the suspect's health. This article elaborates whether the European legal framework, as set by the Council of Europe and the European Union (EU), defines any boundaries for the analytical scope of FDP. After a brief introduction to FDP and a description of the type of data collected through predictive forensic genetics, this article discusses the relevant European legislation and the case law of the European Court of Human Rights (ECtHR) and the Court of Justice of the European Union (CJEU) around privacy, data protection and the use of genetic data. The article attempts to define possible limits for forensic genetic analysis, by eventually trying to predict the jurisprudence of the two European courts.

Population genetic analysis of 12 X-chromosomal STRs in a Swiss sample.

X-chromosomal STRs are a powerful tool to assess a broad variety of complex kinship scenarios. We introduce herewith the first Swiss X-STR dataset based on 1198 individuals (592 female, 606 male), characterized with the Qiagen Investigator® Argus X-12 QS multiplex kit. Anomalous allele patterns, allele and haplotype frequencies, and forensic and population genetic parameters are presented. We detected linkage disequilibrium within three out of the four designated linkage groups and no apparent intra-national population substructure. We compared the dataset to a global panel of X-STR datasets and it fits well in the European context, as expected.

Expanding the Swiss autosomal marker set to 32 STRs.

By genotyping 1198 individuals with the Qiagen Investigator® HDplex Kit, we expand the Swiss autosomal STR dataset to 32 loci, providing additional resources for complex kinship cases. We present the first high-quality allele frequency dataset for loci D2S1360, D5S2500, D7S1517, and D10S2325 that will be accessible through the ENFSI reference database STRidER. For loci D3S1744, D4S2366, D6S474, D8S1132, and D21S2055, we provide a first European STRidER dataset.

Efficient DNA Sampling in Burglary Investigations.

In terms of crime scene investigations by means of forensic DNA-analyses, burglaries are the number one mass crime in Switzerland. Around one third of the DNA trace profiles registered in the Swiss DNA database are related to burglaries. However, during the collection of potential DNA traces within someone's residence after a burglary, it is not known whether the sampled DNA originated from the perpetrator or from an inhabitant of said home. Because of the high incidence of burglaries, crime scene investigators usually do not collect reference samples from all the residents for economical and administrative reasons. Therefore, the presumably high probability that a DNA profile belonging to a person authorized to be at the crime scene ends up being sent to a DNA database for comparison, has to be taken into account. To our knowledge, no investigation has been made to evaluate the percentage of these non-perpetrator profiles straying into DNA databases. To shed light on this question, we collected reference samples from residents who had been victims of recent burglaries in their private homes. By comparing the profiles established from these reference samples with the profiles generated from trace DNA, we can show that the majority of the DNA samples collected in burglary investigations belong to the residents. Despite the limited number of cases included in the study, presumably due to a crime decline caused by the pandemic, we further show that trace DNA collection in the vicinity of the break and entry area, in particular window and door glasses, is most promising for sampling perpetrator instead of inhabitant DNA.

DNA-free does not mean RNA-free-The unwanted persistence of RNA.

Contact shots to the head often provoke a transfer of biological traces into firearm barrels, which are not visible at endoscopic inspection. STR-PCR can amplify these latent traces and assign them to the victim. Via RNA-DNA-co-extraction also miRNA can be detected, which allow a conclusion to be drawn about the body fluid or tissue. Molecular genetic analysis of experimental stains in firearm barrels requires the guarantee that the barrel is initially free of any nucleic acid. Twelve shots were fired to so-called "reference cubes" (10 % gelatine, 12 cm edge length, embedded paint-blood-pad) using three current handguns: from 20 and 30 cm distance, four at close range (1-2.5 cm) and six contact shots. After endoscopic examination and swabbing of the barrels, a previously described mechanical and chemical cleaning using DNAExitusPlus™ was performed. The inner surface of the barrel was thoroughly wiped off using moistened forensic swabs, which were submitted to RNA-DNA-co-extraction. The combined thorough mechanical cleaning with Ballistol® and the application of DNAExitusPlus™ eliminated any profilable DNA in all samples. However, in 10 of 12 samples RNA concentrations between 0.11 - 0.79 ng/µl were measured. Furthermore, in 9 of 12 samples blood-specific miRNA (miR-451a) was detected. Summarizing, none of the experimentally contaminated barrels was RNA-free despite the performed cleaning procedure. Further investigation showed, that even "professional" cleaning by a gunsmith did not remove RNA.

The Y-chromosomal haplotype and haplogroup distribution of modern Switzerland still reflects the alpine divide as a geographical barrier for human migration.

A sample of 606 Swiss individuals has been characterized for 27 Y-STR and 34 Y-SNPs, defining major European haplogroups. For the first time, a subsample from the southernmost part of Switzerland, the Italian speaking canton Ticino, has been included. The data reveals significant intra-national differences in the distribution of haplogroups R1b-U106, R1b-U152, I1 and J2a north and south of the alpine divide, with R1b-U152 being the most frequent haplogroup among all Swiss subpopulations, reaching 26 % in average and 53 % in the Ticino sample. In addition, a high percentage of haplogroup E1b1b-M35 in Eastern Switzerland corresponds well with data reported from Western Austria. In general, we detected a low level of differentiation between the subgroups north of the alpine divide. The dataset also revealed a variety of microvariants. Some of them were previously known to be associated with particular haplogroups. However, we discovered one microvariant in DYS533 that seems to be closely associated with haplogroup I2-P215 (xM223). This association had not yet been reported to date. The concordance study with two STR-kits suggests that the DYS533 microvariant is due to an InDel in the flanking regions of the marker. One individual carried a large deletion, frequently detected in people of East Asian ancestry, encompassing the amelogenin locus. To our knowledge, this is the first time that such a deletion has been observed within European haplogroup R1b-U152. This is the first comprehensive Y chromosomal dataset for Switzerland, demonstrating significant population substructure due to an intra-national geographical barrier.

A "forensic biobank" to establish comprehensive genetic frequency data for Switzerland.

Comparatively little knowledge is available about the genetic structure of the Swiss population. Missing allele frequency data for some markers frequently used in forensics and paternity / kinship testing is just one practical aspect of this lack of genetic data. Therefore, in an attempt to fill this gap, we established a biobank of 1198 Swiss blood samples, systematically collected throughout the whole country. For the first time, this collection contains a population sample from the southernmost Italian speaking canton of Ticino. In this article, we share the experiences gained with the sampling procedure. Furthermore we present autosomal allele frequencies for 23 loci and a concordance check between the two multiplex PCR kits GlobalFiler® and PowerPlex® Fusion 6C. Statistical evaluation of the data revealed only small genetic differences among regional subpopulations and among language subgroups. The autosomal allele frequencies can therefore be used for forensics and paternity / kinship testing as a valid nationwide dataset. Additionally, the informative value of the three Y-STR markers included in the PowerPlex® Fusion 6C kit was assessed. We could demonstrate that the 3-loci-haplotype can be very informative, with an average haplotype frequency in the relevant Western European metapopulation of 0.026.

DNA recovery from gelatin fingerprint lifters by direct proteolytic digestion.

Fingerprints are a valuable source for DNA profiling in forensic investigations. In practice, the fingerprints are routinely visualized first by powder staining and then often transferred to tapes or gelatin lifters for storage or examination. If at all, fingerprints are usually sampled for DNA in a second step. To target the DNA sampling in an optimal way, it is essential to know how much of the DNA in the sample remains in place and how much is transferred to the lifter. In the present study we addressed this question analyzing 16 pairs of thumb prints and revealed that more than 80% of the DNA from a fingerprint is transferred to the gelatin lifter. Therefore, subsequent DNA sampling of the stored gelatin lifters appears more promising than recovery of the residual DNA from the original fingerprint. Furthermore, as a proof of principle, we developed a protocol for the direct extraction of DNA from gelatin fingerprint lifters by proteolytic digestion of the gelatin matrix followed by organic extraction. We show that DNA recovery from gelatin lifters by this direct extraction protocol is more efficient compared to swabbing the lifter followed by standard magnetic bead extraction of swabs. However, given the more elaborate protocol for direct extraction, we would still recommend the swab technique as the method of choice for forensic routine work.

Touch DNA sampling with SceneSafe Fast™ minitapes.

To achieve optimal results in the forensic analysis of trace DNA, choosing the right collection technique is crucial. Three common approaches are currently well-established for DNA retrieval from items of clothing, notably cutting, swabbing and tape-lifting. The latter two are non-destructive and therefore preferable on items of value. Even though the most recently established technique of DNA retrieval by adhesive tapes is widely used since quite some years now, little information has been published so far on how well it performs compared to other methods. Even more important, when it comes to choosing the right DNA extraction method for forensic lifting-tapes, the available information one can rely on as a forensic geneticist is quite scarce. In our study we compared the two widely used, commercially available and automation suitable magnetic bead-based extraction methods "iPrep Forensic Kit" and "PrepFiler Express BTA™ Kit" to conventional organic solvent extraction. The results demonstrate that DNA extraction from standardized saliva samples applied to SceneSafe Fast™ minitapes is most efficient with phenol-chloroform. We also provide evidence that SceneSafe Fast™ minitapes perform better than wet cotton swabs in the sampling of touch DNA from cotton fabric. Applying the tape only once in every spot on the tissue is thereby sufficient for a considerably better collection performance of the tapes compared to swabbing.

STRAF-A convenient online tool for STR data evaluation in forensic genetics.

Population data in forensic genetics has to be checked for a variety of statistical parameters before it can be employed for case work. A lot of very powerful statistical tools are available for this task, most of them developed by labs having their research focus on population genetics or evolution. However, most of these programs require a substantial amount of experience. In addition, to our knowledge, none of the freely available programs calculates all the common parameters for a population study in forensic genetics at once, based on a single input file. We present here a convenient online tool that fills this gap. STRAF (STR Analysis for Forensics) provides an intuitive interface and input file format and computes all the relevant parameters for a classical population study based on autosomal STR data at once and in a convenient way. In addition, STRAF includes a PCA module that can be used for population substructure detection or quality control. The results generated by the program were verified by recalculating parameters from an already published population study.

Electrostatic sampling of trace DNA from clothing.

During acts of physical aggression, offenders frequently come into contact with clothes of the victim, thereby leaving traces of DNA-bearing biological material on the garments. Since tape-lifting and swabbing, the currently established methods for non-destructive trace DNA sampling from clothing, both have their shortcomings in collection efficiency and handling, we thought about a new collection method for these challenging samples. Testing two readily available electrostatic devices for their potential to sample biological material from garments made of different fabrics, we found one of them, the electrostatic dust print lifter (DPL), to perform comparable to well-established sampling with wet cotton swabs. In simulated aggression scenarios, we had the same success rate for the establishment of single aggressor profiles, suitable for database submission, with both the DPL and wet swabbing. However, we lost a substantial amount of information with electrostatic sampling, since almost no mixed aggressor-victim profiles suitable for database entry could be established, compared to conventional swabbing. This study serves as a proof of principle for electrostatic DNA sampling from items of clothing. The technique still requires optimization before it might be used in real casework. But we are confident that in the future it could be an efficient and convenient contribution to the toolbox of forensic practitioners.

About DNA databasing and investigative genetic analysis of externally visible characteristics: A public survey.

During the last decade, DNA profiling and the use of DNA databases have become two of the most employed instruments of police investigations. This very rapid establishment of forensic genetics is yet far from being complete. In the last few years novel types of analyses have been presented to describe phenotypically a possible perpetrator. We conducted the present study among German speaking Swiss residents for two main reasons: firstly, we aimed at getting an impression of the public awareness and acceptance of the Swiss DNA database and the perception of a hypothetical DNA database containing all Swiss residents. Secondly, we wanted to get a broader picture of how people that are not working in the field of forensic genetics think about legal permission to establish phenotypic descriptions of alleged criminals by genetic means. Even though a significant number of study participants did not even know about the existence of the Swiss DNA database, its acceptance appears to be very high. Generally our results suggest that the current forensic use of DNA profiling is considered highly trustworthy. However, the acceptance of a hypothetical universal database would be only as low as about 30% among the 284 respondents to our study, mostly because people are concerned about the security of their genetic data, their privacy or a possible risk of abuse of such a database. Concerning the genetic analysis of externally visible characteristics and biogeographical ancestry, we discover a high degree of acceptance. The acceptance decreases slightly when precise characteristics are presented to the participants in detail. About half of the respondents would be in favor of the moderate use of physical traits analyses only for serious crimes threatening life, health or sexual integrity. The possible risk of discrimination and reinforcement of racism, as discussed by scholars from anthropology, bioethics, law, philosophy and sociology, is mentioned less frequently by the study participants than we would have expected. A national DNA database and the widespread use of DNA analyses for police and justice have an impact on the entire society. Therefore the concerns of lay persons from the respective population should be heard and considered. The aims of this study were to draw a broader picture of the public opinion on DNA databasing and to contribute to the debate about the possible future use of genetics to reveal phenotypic characteristics. Our data might provide an additional perspective for experts involved in regulatory or legislative processes.