Sequencing and de novo assembly of 150 genomes from Denmark as a population reference.

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.

Statistical Modelling Investigation of MALDI-MSI-Based Approaches for Document Examination.

Questioned document examination aims to assess if a document of interest has been forged. Spectroscopy-based methods are the gold standard for this type of evaluation. In the past 15 years, Matrix-Assisted Laser Desorption Ionisation-Mass Spectrometry Imaging (MALDI-MSI) has emerged as a powerful analytical tool for the examination of finger marks, blood, and hair. Therefore, this study intended to explore the possibility of expanding the forensic versatility of this technique through its application to questioned documents. Specifically, a combination of MALDI-MSI and chemometric approaches was investigated for the differentiation of seven gel pens, through their ink composition, over 44 days to assess: (i) the ability of MALDI MSI to detect and image ink chemical composition and (ii) the robustness of the combined approach for the classification of different pens over time. The training data were modelled using elastic net logistic regression to obtain probabilities for each pen class and assess the time effect on the ink. This strategy led the classification model to yield predictions matching the ground truth. This model was validated using signatures generated by different pens (blind to the analyst), yielding a 100% accuracy in machine learning cross-validation. These data indicate that the coupling of MALDI-MSI with machine learning was robust for ink discrimination within the dataset and conditions investigated, which justifies further studies, including that of confounders such as paper brands and environmental factors.

Asphyxia homicides in Denmark 1992-2016.

In this retrospective study , we present the findings in 250 homicides by asphyxia in Denmark in a 25-year period, with a particular focus on the autopsy findings in strangulation. Our intention is for the results to be used in future death investigations, where difficulties in interpretation of findings in potential asphyxial deaths arise. Asphyxia homicides showed a strong bias with respect to sex, age, and homicide type. The frequent female victim was typically an adult, whereas the rarer male victim was most often a child. Female offenders most often killed their children, and male offenders most often killed their female partner. Generally, most asphyxia homicides took place in a domestic setting. Manual strangulation and ligature strangulation were the most common mechanisms of asphyxia homicides (81.6%). A lack of petechial hemorrhages, especially in the conjunctiva, was rare in homicidal strangulation, but there were exceptions, especially when there was postmortem decomposition, making it impossible to verify them. Most victims of strangulation had skin lesions in the face (including the jawline) or on the neck, with accompanying hemorrhages in muscle and connective tissue, but the findings could be subtle or compounded by decomposition. Fractures of the laryngo-hyoid complex were common in strangulation, particularly in manual strangulation (chi-sq = 4.0993, df = 1, P < 0.05) and were clearly related to the age of the victim (chi-sq = 82.193, df = 4, P < 0.001). In children and young adults dying from homicidal strangulation, a lack of fractures is to be expected, while a lack of fractures is unusual, but not entirely unexpected, for adults and aged people.

Assessment of XCMS Optimization Methods with Machine-Learning Performance.

The metabolomics field is under rapid development. In particular, biomarker identification and pathway analysis are growing, as untargeted metabolomics is usable for discovery research. Frequently, new processing and statistical strategies are proposed to accommodate the increasing demand for robust and standardized data. One such algorithm is XCMS, which processes raw data into integrated peaks. Multiple studies have tried to assess the effect of optimizing XCMS parameters, but it is challenging to quantify the quality of the XCMS output. In this study, we investigate the effect of two automated optimization tools (Autotuner and isotopologue parameter optimization (IPO)) using the prediction power of machine learning as a proxy for the quality of the data set. We show that optimized parameters outperform default XCMS settings and that manually chosen parameters by liquid chromatography-mass spectrometry (LC-MS) experts remain the best. Finally, the machine-learning approach of quality assessment is proposed for future evaluations of newly developed optimization methods because its performance directly measures the retained signal upon preprocessing.

Gunshot homicides in Denmark 1992-2016.

The majority of homicides in the world are caused by firearms and a good understanding of gunshot wounds is important for the forensic pathologist. We have analyzed all 315 homicides by gunshots in Denmark during 1992-2016 with focus on information relevant to forensic pathologists. Of the 1417 homicides in Denmark in 1992-2016, 315 (22.2%) had gunshot trauma as the primary homicide method. During 1992-2016, there has been a significant decrease in the number of gunshot homicides overall, of homicides committed with hunting weapons, and of domestic gunshot homicides. Most victims (70.5%) and offenders (93.7% of homicides with known offenders) were males. Male victims were significantly younger than female victims. Most victims were killed with handguns (43.8%) or shotguns (41.0%). Compared to all other weapons, the victims killed with shotguns had fewer entry wounds, a higher New Injury Severity Score (NISS), and a higher proportion of contact/close-range entry wounds. The head (58.1%) and thorax (46.7%) were often injured, with injuries to the head being more common in contact/close-range entry wounds. The results show that gunshot injuries in homicides are different from suicides and accidents and clearly are affected by the homicide type, the sex of the victim, and the weapon used and that these entities are intertwined. Our findings provide an evidence-based foundation for use in death investigations and for policy development in the area of interpersonal violence.

Assembly and analysis of 100 full MHC haplotypes from the Danish population.

Genes in the major histocompatibility complex (MHC, also known as HLA) play a critical role in the immune response and variation within the extended 4-Mb region shows association with major risks of many diseases. Yet, deciphering the underlying causes of these associations is difficult because the MHC is the most polymorphic region of the genome with a complex linkage disequilibrium structure. Here, we reconstruct full MHC haplotypes from de novo assembled trios without relying on a reference genome and perform evolutionary analyses. We report 100 full MHC haplotypes and call a large set of structural variants in the regions for future use in imputation with GWAS data. We also present the first complete analysis of the recombination landscape in the entire region and show how balancing selection at classical genes have linked effects on the frequency of variants throughout the region.

Improved medical treatment could explain a decrease in homicides with a single stab wound.

Since the 1990s, there has been a reduction in the homicide rate in Denmark and other Western countries. Our hypothesis is that part of the decrease in the sharp force homicide rate can be explained by better and faster medical treatment over time, and we explore this via stab wound homicides, the largest group of homicides in Denmark. To investigate our hypothesis we undertook an epidemiological study of 428 stab wound homicides in Denmark 1992-2016 based on autopsy reports with registration of stab wounds, quantification of injury severity, treatment intensity and survival time. During 1992-2016, there was a significant reduction in the annual number of victims with a single stab wound, but no reduction in victims with multiple stab wounds. Victims with single stab wounds reached the hospital more often, survived longer and had less severe injuries (New Injury Severity Score (NISS)) than victims with multiple stab wounds. Higher NISS correlated with shorter survival time for all the stab wound victims and for the subgroup that underwent medical treatment. During the 25-year study period, the proportion of victims who underwent surgery before dying increased threefold. The victims in the first half of the study period had shorter survival times than the victims in the last half. We concluded that better and faster medical treatment could partly be responsible for the observed decrease in the number of single stab wound homicides and thereby possibly also in the total number of stab wound homicides.

Whole-Genome Sequencing of Aggregatibacter Species Isolated from Human Clinical Specimens and Description of Aggregatibacter kilianii sp. nov.

Aggregatibacter species are commensal bacteria of human mucosal surfaces that are sometimes involved in serious invasive infections. During the investigation of strains cultured from various clinical specimens, we encountered a coherent group of 10 isolates that could not be allocated to any validly named species by phenotype, mass spectrometry, or partial 16S rRNA gene sequencing. Whole-genome sequencing revealed a phylogenetic cluster related to but separate from Aggregatibacter aphrophilus The mean in silico DNA hybridization value for strains of the new cluster versus A. aphrophilus was 56% (range, 53.7 to 58.0%), whereas the average nucleotide identity was 94.4% (range, 93.9 to 94.8%). The new cluster exhibited aggregative properties typical of the genus Aggregatibacter Key phenotypic tests for discrimination of the new cluster from validly named Aggregatibacter species are alanine-phenylalanine-proline arylamidase, N-acetylglucosamine, and ß-galactosidase. The name Aggregatibacter kilianii is proposed, with PN_528 (CCUG 70536T or DSM 105094T) as the type strain.

Nature and nurture: a case of transcending haematological pre-malignancies in a pair of monozygotic twins adding possible clues on the pathogenesis of B-cell proliferations.

We describe a comprehensive molecular analysis of a pair of monozygotic twins, who came to our attention when one experienced amaurosis fugax and was diagnosed with JAK2+ polycythaemia vera. He (Twin A) was also found to have an asymptomatic B-cell chronic lymphocytic leukaemia (B-CLL). Although JAK2-, Twin B was subsequently shown to have a benign monoclonal B-cell lymphocytosis (MBL). Flow cytometric and molecular analyses of the B-cell compartments revealed different immunoglobulin light and heavy chain usage in each twin. We hypothesized that whole exome sequencing could help delineating the pattern of germline B-cell disorder susceptibility and reveal somatic mutations potentially contributing to the differential patterns of pre-malignancy. Comparing bone marrow cells and T cells and employing in-house engineered integrative analysis, we found aberrations in Twin A consistent with a myeloid neoplasm, i.e. in TET2, RUNX1, PLCB1 and ELF4. Employing the method for detecting high-ranking variants by extensive annotation and relevance scoring, we also identified shared germline variants in genes of proteins interacting with B-cell receptor signalling mediators and the WNT-pathway, including IRF8, PTPRO, BCL9L, SIT1 and SIRPB1, all with possible implications in B-cell proliferation. Similar patterns of IGHV-gene usage to those demonstrated here have been observed in inherited acute lymphoblastic leukaemia. Collectively, these findings may help in facilitating identification of putative master gene(s) involved in B-cell proliferations in general and MBL and B-CLL in particular.

Automatic removal of soft tissue from 3D dental photo scans; an important step in automating future forensic odontology identification.

The potential of intraoral 3D photo scans in forensic odontology identification remains largely unexplored, even though the high degree of detail could allow automated comparison of ante mortem and post mortem dentitions. Differences in soft tissue conditions between ante- and post mortem intraoral 3D photo scans may cause ambiguous variation, burdening the potential automation of the matching process and underlining the need for limiting inclusion of soft tissue in dental comparison. The soft tissue removal must be able to handle dental arches with missing teeth, and intraoral 3D photo scans not originating from plaster models. To address these challenges, we have developed the grid-cutting method. The method is customisable, allowing fine-grained analysis using a small grid size and adaptation of how much of the soft tissues are excluded from the cropped dental scan. When tested on 66 dental scans, the grid-cutting method was able to limit the amount of soft tissue without removing any teeth in 63/66 dental scans. The remaining 3 dental scans had partly erupted third molars (wisdom teeth) which were removed by the grid-cutting method. Overall, the grid-cutting method represents an important step towards automating the matching process in forensic odontology identification using intraoral 3D photo scans.

Endogenous retroviruses and multiple sclerosis-new pieces to the puzzle.

The possibility that retroviruses play a role in multiple sclerosis (MS) has long been considered; accumulating findings suggest this to be most likely in the form of human endogenous retroviruses (HERVs). A genetic test series of fifty endogenous retroviral loci for association with MS in Danes showed SNP markers near a specific endogenous retroviral locus, HERV-Fc1 located on the X-chromosome, to be positive. Bout Onset MS was associated with the HERV-Fc1 locus, while a rarer form, Primary Progressive MS, was not. Moreover, HERV-Fc1 Gag RNA in plasma was increased 4-fold in patients with recent history of attacks, relative to patients in a stable state and to healthy controls.Finally, genetic variations in restriction genes for retroviruses influence the risk of MS, providing further support for a role of retroviral elements in disease.We speculate that endogenous retroviruses may activate the innate immune system in a variety of ways, involving the host proteins, TRIMs, TLRs, TREXs and STING. Observations in HIV-positive patients suggest that antiretroviral drugs can curb MS. Thus, these new findings regarding the etiology and pathogenesis of MS, suggest alternative ways to challenge autoimmune diseases.

Intimate partner homicides in Denmark 1992-2016.

Intimate partner homicide is one of the most common types of homicide and a significant contributor to domestic homicides worldwide, especially affecting females. We focus on the intimate partner homicides in Denmark during 1992-2016. Though gender identity data was unavailable, sex data from official documents enabled critical analysis. Of the 1417 homicides in the period, 26.5% were intimate partner homicides, i.e., 55.6% of female and 8.9% of male victims. The annual intimate partner homicide rate was 0.28 per 100,000 (0.44 for female victims and 0.12 for male victims), declining at a lower rate than other types of homicide. Most victims of intimate partner homicides were females (79.3%). The demographics of the victims and the characteristics of the homicides were markedly different depending on victim sex. Female victims were killed by more varied methods, with more severe injuries and followed by suicide in 26.5% and with multiple homicide victims in 8.1%.

Large-Scale metabolomics: Predicting biological age using 10,133 routine untargeted LC-MS measurements.

Untargeted metabolomics is the study of all detectable small molecules, and in geroscience, metabolomics has shown great potential to describe the biological age-a complex trait impacted by many factors. Unfortunately, the sample sizes are often insufficient to achieve sufficient power and minimize potential biases caused by, for example, demographic factors. In this study, we present the analysis of biological age in ~10,000 toxicologic routine blood measurements. The untargeted screening samples obtained from ultra-high pressure liquid chromatography-quadruple time of flight mass spectrometry (UHPLC- QTOF) cover + 300 batches and + 30 months, lack pooled quality controls, lack controlled sample collection, and has previously only been used in small-scale studies. To overcome experimental effects, we developed and tested a custom neural network model and compared it with existing prediction methods. Overall, the neural network was able to predict the chronological age with an rmse of 5.88 years (r2  = 0.63) improving upon the 6.15 years achieved by existing normalization methods. We used the feature importance algorithm, Shapley Additive exPlanations (SHAP), to identify compounds related to the biological age. Most importantly, the model returned known aging markers such as kynurenine, indole-3-aldehyde, and acylcarnitines along with a potential novel aging marker, cyclo (leu-pro). Our results validate the association of tryptophan and acylcarnitine metabolism to aging in a highly uncontrolled large-s cale sample. Also, we have shown that by using robust computational methods it is possible to deploy large LC-MS datasets for metabolomics studies to reduce the risk of bias and empower aging studies.

Blunt force homicides in Denmark 1992-2016.

Blunt force trauma is a common homicide method, inflicted in three different ways: bodily force, assault with blunt objects of various types and falls from height. The objective of this study is to provide thorough information on blunt force homicides with data on the victims, the offenders, the surrounding circumstances, the injury methods, the extent of injuries, and survival time, which will help inform the inexperienced as well as the seasoned forensic pathologist in their daily work with death investigation and as expert witnesses in court. We have analyzed autopsy reports and available case files of 311 blunt force homicides, making up 21.9% of all homicides in Denmark during 1992-2016. Most victims and offenders were male. Altercation in the setting of nightlife and intoxication was common in male victims, while most female victims were killed in a domestic setting. Bodily force was the most common primary homicide method, followed by assault with a blunt object and fall from height. The head was the region that most often had external injuries, with no noteworthy difference between cases with bodily force and blunt objects. Two out of three victims had one or more lacerations, most often located on the head and more often on the front. Brain injury was the primary cause of death in at least 72.0% victims. Compared to bodily force victims of blunt object assault were especially prone to skull and brain injuries, had a higher trauma score, and more died at the crime scene and had a shorter survival time.

From genotype to phenotype: Genetic redundancy and the maintenance of an adaptive polymorphism in the context of high gene flow.

A central question in evolution is how several adaptive phenotypes are maintained within a species. Theory predicts that the genetic determination of a trait, and in particular the amounts of redundancy in the mapping of genotypes to phenotypes, mediates evolutionary outcomes of phenotypic selection. In Mediterranean wild thyme, numerous discrete chemical phenotypes (chemotypes) occur in close geographic proximity. Chemotypes are defined by the predominant monoterpene produced by individual plants in their essential oil. In this study, we analyze the ecological genetics of six chemotypes nested within two well-established chemical families (hereafter ecotypes). Ecotypes, and chemotypes within ecotypes, are spatially segregated, and their distributions track local differences in the abiotic environment. By combining population genomic, phenotypic, and environmental data from 700 individuals, we show how the genetics of ecotype determination mediates this evolutionary response. Variation in three terpene-synthase loci explains variation in ecotype identity, with one single locus accounting for as much as 78% of this variation. Phenotypic selection combined with low segregating genotypic redundancy of ecotypes leaves a clear footprint at the genomic level: alleles associated with ecotype identity track environmental variation despite extensive gene flow. Different chemotypes within each ecotype differentially track environmental variation. Their identity is determined by multiple loci and displays a wider range of genotypic redundancy that dilutes phenotypic selection on their characteristic alleles. Our study thus provides a novel illustration of how genetic redundancy of a phenotype modulates the ability of selection to maintain adaptive differentiation. Identifying the precise genetics of the chemical polymorphism in thyme is the next crucial step for our understanding of the origin and maintenance of a polymorphism that is present in many aromatic plants.

A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

Frequent genomic loss at chr16p13.2 is associated with poor prognosis in colorectal cancer.

Genomic alterations play important roles in colorectal cancer (CRC) carcinogenesis. Here, we aimed to identify and characterize recurrent copy-number alterations (CNAs) associated with clinical outcome of CRC by the use of single nucleotide polymorphism arrays, genomic quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). Colorectal neoplasia specimens and paired germline samples from 144 patients (40 adenomas and 104 carcinomas) as well as 40 CRC cell lines were investigated. This large dataset revealed frequent loss, including homozygous loss, at chr16p13.2 (from 5.9 to 7.42Mb). The loss was observed in 30% of adenomas and even more frequently in carcinomas, 56%, indicating that the loss define a subset of adenomas with a propensity for invasion. Consistent with this, the loss occurred twice as frequent in villous (40%) as in tubular adenomas (20%). The loss occurred independently of microsatellite stability and could be validated by qPCR in an independent sample cohort (n = 71). In Stage II/III, microsatellite stable (MSS) CRC it was associated with poor recurrence free survival (hazard ratio 2.4; p = 0.02; Multivariate Cox regression analysis). No transcriptional consequences of the losses were observed, and the only gene, A2BP1, located in the region showed no mutations. Correlation with other CNAs was established for chr3p22 in carcinomas and chr20p (inverse) in adenomas. FISH documented the chr16p13.2 region to be involved in complex structural rearrangements that included translocation to chr3p22 in some cases. The findings indicate that structural rearrangements involving chr16p13.2 are very frequent in colorectal neoplasia, often lead to homozygous deletion, and are associated with poor clinical outcome.

Hybrid lentivirus-transposon vectors with a random integration profile in human cells.

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans directs efficient transposon mobilization from DNA circles in vector-transduced cells. Both transfected plasmid DNA and transduced IDLVs can serve as the source of active transposase. Most important, we demonstrate that the SB transposase overrides the natural lentiviral integration pathway and directs vector integration less frequently toward transcriptional units, resulting in a random genomic integration profile. The novel hybrid vector system combines the attractive features of efficient gene delivery by viral transduction and a safer genomic integration profile by DNA transposition.

Sharp Force Homicide in Denmark 1992-2016.

Sharp force trauma is a common homicide method. The weapon is typically a knife, which is easily accessible and does not require special skills. We have analyzed all 471 sharp force homicides in Denmark during 1992-2016 with special focus on aspects that are relevant to forensic pathologists, including the distribution of wounds and organ injuries. Most homicides were committed inside with a kitchen knife. The front left thorax was the most common area to be affected by sharp force trauma. In 18.9% of the victims, there was only one sharp injury, the majority on the thorax. The most common trajectory for stab wounds was directly posterior with no deviation to the sides or up/down followed by directly anterior. The heart (including pericardium) and lungs (including hemo- and pneumothorax) had injuries in more than 75% of the victims. 67% of victims were males. Female victims had more sharp force injuries and defense wounds than male victims. Most females were killed in domestic homicides (73.7%), most commonly in partner killings (56.4%). In contrast, many male victims were killed in a setting of nightlife/intoxication (34.0%) most by a friend/acquaintance delivering a few stab wounds. The results clearly show strong sex differences in both victims and offenders. This could be useful for shaping policies and public opinion, and as a route for understanding the developments in interpersonal violence. In the narrow setting of death investigation, our results will provide an evidence-based approach to understanding the injury patterns in sharp force homicide.

Homicide in Denmark 1992-2016.

We present the findings for homicides in Denmark for 1992-2016. There were 1417 homicide victims (62.2% males, 37.8% females) that were killed in 1321 homicide events. The most common methods were sharp force trauma (33.2%), gunshot (22.2%), blunt force trauma (21.9%) and asphyxia (17.6%), and all methods exhibited a reduction during the study period. The homicide rate was 1.05 per 100,000, 1.32 per 100,000 for males, and 0.78 per 100,000 for females. Domestic homicides were the largest main group of homicides (76.5% of all female victims vs. 23.6% of male victims). Of the non-domestic homicides, 84.2% of victims were male, the largest group being in the setting of nightlife and/or intoxication. Most female victims (76.9%) were killed by someone in their family, while the largest share of male victims (34.5%) were killed by a friend or acquaintance. The offenders were males in 87.9% of all homicides.