Rescue of Mycobacterium bovis DNA Obtained from Cultured Samples during Official Surveillance of Animal TB: Key Steps for Robust Whole Genome Sequence Data Generation.

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention.

Genetic investigations of 100 inherited cardiac disease-related genes in deceased individuals with schizophrenia.

Cardiac diseases and sudden cardiac death (SCD) are more prevalent in individuals diagnosed with schizophrenia compared to the general population, with especially coronary artery disease (CAD) as the major cardiovascular cause of death. Antipsychotic medications, genetics, and lifestyle factors may contribute to the increased SCD in individuals with schizophrenia. The role of antipsychotic medications and lifestyle factors have been widely investigated, while the genetic predisposition to inherited cardiac diseases in schizophrenia is poorly understood. In this study, we examined 100 genes associated with inherited cardiomyopathies and cardiac channelopathies in 97 deceased individuals diagnosed with schizophrenia for the prevalence of genetic variants associated with SCD. The deceased individuals had various causes of death and were included in the SURVIVE project, a prospective, autopsy-based study of mentally ill individuals in Denmark. This is the first study of multiple inherited cardiac disease-related genes in deceased individuals with diagnosed schizophrenia to shed light on the genetic predisposition to SCD in individuals with schizophrenia. We found no evidence for an overrepresentation of rare variants with high penetrance in inherited cardiac diseases, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) consensus guidelines. However, we found that the deceased individuals had a statistically significantly increased polygenic burden caused by variants in the investigated heart genes compared to the general population. This indicates that common variants with smaller effects in heart genes may play a role in schizophrenia.

Targeted exon sequencing in deceased schizophrenia patients in Denmark.

Schizophrenia patients have higher mortality rates and lower life expectancy than the general population. However, forensic investigations of their deaths often fail to determine the cause of death, hindering prevention. As schizophrenia is a highly heritable condition and given recent advances in our understanding of the genetics of schizophrenia, it is now possible to investigate how genetic factors may contribute to mortality. We made use of findings from genome-wide association studies (GWAS) to design a targeted panel (PsychPlex) for sequencing of exons of 451 genes near index single nucleotide polymorphisms (SNPs) identified with GWAS. We sequenced the DNA of 95 deceased schizophrenia patients included in SURVIVE, a prospective, autopsy-based study of mentally ill persons in Denmark. We compared the allele frequencies of 1039 SNPs in these cases with the frequencies of 2000 Danes without psychiatric diseases and calculated their deleteriousness (CADD) scores. For 81 SNPs highly associated with schizophrenia and CADD scores above 15, expression profiles in the Genotype-Tissue Expression (GTEx) Project indicated that these variants were in exons, whose expressions are increased in several types of brain tissues, particularly in the cerebellum. Molecular pathway analysis indicated the involvement of 163 different pathways. As for rare SNP variants, most variants were scored as either benign or likely benign with an average of 17 variants of unknown significance per individual and no pathogenic variant. Our results highlight the potential of DNA sequencing of an exon panel to discover genetic factors that may be involved in the development of schizophrenia.

Cartilaginous fishes offer unique insights into the evolution of the nuclear receptor gene repertoire in gnathostomes.

Nuclear receptors (NRs) are key transcription factors that originated in the common ancestor of metazoans. The vast majority of NRs are triggered by binding to either endogenous (e.g. retinoic acid) or exogenous (e.g. xenobiotics) ligands, and their evolution and expansion is tightly linked to the function of endocrine systems. Importantly, they represent classic targets of physiological exploitation by endocrine disrupting chemicals. The NR gene repertoire in different lineages has been shaped by gene loss, duplication and mutation, denoting a dynamic evolutionary route. As the earliest diverging class of gnathostomes (jawed vertebrates), cartilaginous fishes offer an exceptional opportunity to address the early diversification of NR gene families and the evolution of the endocrine system in jawed vertebrates. Here we provide an exhaustive analysis into the NR gene composition in five elasmobranch (sharks and rays) and two holocephalan (chimaeras) species. For this purpose, we generated also a low coverage draft genome assembly of the chimaera small-eyed rabbitfish, Hydrolagus affinis. We show that cartilaginous fish retain an archetypal NR gene repertoire, similar to that of mammals and coincident with the two rounds of whole genome duplication that occurred in the gnathostome ancestor. Furthermore, novel gene members of the non-canonical NR0B receptors were found in the genomes of this lineage. Our findings provide an essential view into the early diversification of NRs in gnathostomes, paving the way for functional studies.

Increasing the reference populations for the 55 AISNP panel: the need and benefits.

Ancestry inference for an individual can only be as good as the reference populations with allele frequency data on the SNPs being used. If the most relevant ancestral population(s) does not have data available for the SNPs studied, then analyses based on DNA evidence may indicate a quite distantly related population, albeit one among the more closely related of the existing reference populations. We have added reference population allele frequencies for 14 additional population samples (with >1100 individuals studied) to the 125 population samples previously published for the Kidd Lab 55 AISNP panel. Allele frequencies are now publicly available for all 55 SNPs in ALFRED and FROG-kb for a total of 139 population samples. This Kidd Lab panel of 55 ancestry informative SNPs has been incorporated in commercial kits by both ThermoFisher Scientific and Illumina for massively parallel sequencing. Researchers employing those kits will find the enhanced set of reference populations useful.

Complete mitochondrial DNA sequence of the endangered giant sable antelope (Hippotragus niger variani): insights into conservation and taxonomy.

The giant sable antelope is one of the most endangered African bovids. Populations of this iconic animal, the national symbol of Angola, were recently rediscovered, after many decades of presumed extinction. Even so, their numbers are scarce and hence conservation plans are essential. However, fundamental information such as its taxonomic position, time of divergence and degree of genetic variation are still lacking. Here, we used a museum preserved horn as a source of DNA to describe, for the first time, the complete mitochondrial genome of the giant sable antelope, and provide insights into its evolutionary history. Reads generated by shotgun sequencing were mapped against the mitochondrial genome of common sable antelope and the nuclear genomes of cow and sheep. Phylogenetic reconstruction and divergence time estimate give support to the monophyly of the giant sable and a maximum divergence time of 170 thousand years to the closest subspecies. About 7% of the nuclear genome was mapped against the reference. The genetic resources reported here are now available for future work in the field of conservation genetics and phylogeny, in this and related species.

Population structure and history of Mycobacterium bovis European 3 clonal complex reveal transmission across ecological corridors of unrecognized importance in Portugal.

Mycobacterium bovis causes animal tuberculosis in livestock and wildlife, with an impact on animal health and production, wildlife management, and public health. In this work, we sampled a multi-host tuberculosis community from the official hotspot risk area of Portugal over 16 years, generating the largest available data set in the country. Using phylogenetic and ecological modeling, we aimed to reconstruct the history of circulating lineages across the livestock-wildlife interface to inform intervention and the implementation of genomic surveillance within the official eradication plan. We find evidence for the co-circulation of M. bovis European 1 (Eu1), Eu2, and Eu3 clonal complexes, with Eu3 providing sufficient temporal signal for further phylogenetic investigation. The Eu3 most recent common ancestor (bovine) was dated in the 1990s, subsequently transitioning to wildlife (red deer and wild boar). Isolate clustering based on sample metadata was used to inform phylogenetic inference, unravelng frequent transmission between two clusters that represent an ecological corridor of previously unrecognized importance in Portugal. The latter was associated with transmission at the livestock-wildlife interface toward locations with higher temperature and precipitation, lower agriculture and road density, and lower host densities. This is the first analysis of M. bovis Eu3 complex in Iberia, shedding light on background ecological factors underlying long-term transmission and informing where efforts could be focused within the larger hotspot risk area of Portugal. IMPORTANCE: Efforts to strengthen surveillance and control of animal tuberculosis (TB) are ongoing worlwide. Here, we developed an eco-phylodynamic framework based on discrete phylogenetic approaches informed by M. bovis whole-genome sequence data representing a multi-host transmission system at the livestock-wildlife interface, within a rich ecological landscape in Portugal, to understand transmission processes and translate this knowledge into disease management benefits. We find evidence for the co-circulation of several M. bovis clades, with frequent transmission of the Eu3 lineage among cattle and wildlife populations. Most transition events between different ecological settings took place toward host, climate and land use gradients, underscoring animal TB expansion and a potential corridor of unrecognized importance for M. bovis maintenance. Results stress that animal TB is an established wildlife disease without ecological barriers, showing that control measures in place are insufficient to prevent long-distance transmission and spillover across multi-host communities, demanding new interventions targeting livestock-wildlife interactions.

The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation.

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to leap to the next level.

Endoscopic assessment and grading of Barrett's esophagus using magnification endoscopy and narrow band imaging: impact of structured learning and experience on the accuracy of the Amsterdam classification system.

OBJECTIVE: Several classification systems have been launched to characterize Barrett's esophagus (BE) mucosa using magnification endoscopy with narrow band imaging (ME-NBI). The good accuracy and interobserver agreement described in the early reports were not reproduced subsequently. Recently, we reported somewhat higher accuracy of the classification developed by the Amsterdam group. The critical question then formulated was whether a structured learning program and the level of experience would affect the clinical usefulness of this classification. MATERIAL & METHODS: Two hundred and nine videos were prospectively captured from patients with BE using ME-NBI. From these, 70 were randomly selected and evaluated by six endoscopists with different levels of expertise, using a dedicated software application. First, an educational set was studied. Thereafter, the 70 test videos were evaluated. After classification of each video, the respective histological feedback was automatically given. RESULTS: Within the learning process, there was a decrease in the time needed for evaluation and an increase in the certainty of prediction. The accuracy did not increase with the learning process. The sensitivity for detection of intestinal metaplasia ranged between 39% and 57%, and for neoplasia between 62% and 90%, irrespective of assessor's expertise. The kappa coefficient for the interobserver agreement ranged from 0.25 to 0.30 for intestinal metaplasia, and from 0.39 to 0.48 for neoplasia. CONCLUSION: Using a dedicated learning program, the ME-NBI Amsterdam classification system is suboptimal in terms of accuracy and inter- and intraobserver agreements. These results reiterate the questionable utility of corresponding classification system in clinical routine practice.

Metabolic signature of the pathogenic 22q11.2 deletion identifies carriers and provides insight into systemic dysregulation.

Large deletions at chromosome 22q11.2 are known to cause severe clinical conditions collectively known as 22q11.2 deletion syndrome. Notwithstanding the pathogenicity of these deletions, affected individuals are typically diagnosed in late childhood or early adolescence, and little is known of the molecular signaling cascades and biological consequences immediately downstream of the deleted genes. Here, we used targeted metabolomics to compare neonatal dried blood spot samples from 203 individuals clinically identified as carriers of a deletion at chromosome 22q11.2 with 203 unaffected individuals. A total of 173 metabolites were successfully identified and used to inform on systemic dysregulation caused by the genomic lesion and to discriminate carriers from non-carriers. We found 84 metabolites to be differentially abundant between carriers and non-carriers of the 22q11.2 deletion. A predictive model based on all 173 metabolites achieved high Accuracy (89%), Area Under the Curve (93%), F1 (88%), Positive Predictive Value (94%), and Negative Predictive Value (84%) with tyrosine and proline having the highest individual contributions to the model as well as the highest interaction strength. Targeted metabolomics provides insight into the molecular consequences possibly contributing to the pathology underlying the clinical manifestations of the 22q11 deletion and is an easily applicable approach to first-pass screening for carrier status of the 22q11 to prompt subsequent verification of the genomic diagnosis.

Associations between patterns in comorbid diagnostic trajectories of individuals with schizophrenia and etiological factors.

Schizophrenia is a heterogeneous disorder, exhibiting variability in presentation and outcomes that complicate treatment and recovery. To explore this heterogeneity, we leverage the comprehensive Danish health registries to conduct a prospective, longitudinal study from birth of 5432 individuals who would ultimately be diagnosed with schizophrenia, building individual trajectories that represent sequences of comorbid diagnoses, and describing patterns in the individual-level variability. We show that psychiatric comorbidity is prevalent among individuals with schizophrenia (82%) and multi-morbidity occur more frequently in specific, time-ordered pairs. Three latent factors capture 79% of variation in longitudinal comorbidity and broadly relate to the number of co-occurring diagnoses, the presence of child versus adult comorbidities and substance abuse. Clustering of the factor scores revealed five stable clusters of individuals, associated with specific risk factors and outcomes. The presentation and course of schizophrenia may be associated with heterogeneity in etiological factors including family history of mental disorders.

Declining genetic diversity of European honeybees along the twentieth century.

The European honeybee (Apis mellifera) is a key pollinator and has in the last decades suffered significant population decline. A combination of factors, including decrease in genetic diversity and introduction of Varroa mites, have been suggested to be responsible for these losses, but no definitive cause has yet been appointed. In Europe not only have wild colonies been severely affected, but managed hives have had a massive decline in numbers. To test the hypothesis that honeybees' genetic diversity has decreased in the recent past, we used reduced representation genome sequencing of 40 historical honeybee specimens collected in Natural History collections across Europe and compared them to genomic data from 40 individuals from extant populations (collected post 2006). Our results are consistent with the existence of five evolutionary lineages as previously described, and show a decrease in genetic diversity between historical and extant individuals of the same lineage, as well as high levels of admixture in historical specimens. Our data confirm that a loss of genetic diversity has occurred during the last century, potentially increasing honeybees' vulnerability to contemporary ecological and anthropogenic stressors.

Frequencies of HID-ion ampliseq ancestry panel markers among greenlanders.

The HID-Ion AmpliSeq Ancestry Panel from Life Techologies includes 123 SNPs from the Seldin panel and 55 SNPs from Kidd panel in a single multiplex assay that helps to determine the continental biogeographic ancestry of individuals. We tested the panel on 104 Greenlanders, divided into a training set of 89 individuals and a test set of 15 individuals. All loci showed genotype distributions consistent with Hardy-Weinberg expectations. Linkage disequilibrium tests indicated that 14 pairs of loci were in association in Greenlanders. Population assignment of the training set to populations included in the HID SNP genotyper plugin placed most individuals in American, Asian, and in a few cases European populations. By including the genotype frequencies of this training set as a possible population of origin, all 15 individuals from the test set were correctly predicted to be Greenlanders using the Seldin SNPs, and nine were classified as Greenlanders using the Kidd SNPs. Population structure analysis indicated that Greenlanders have a genetic profile that is distinguishable from those of populations from America or Asia.

Next-generation biology: Sequencing and data analysis approaches for non-model organisms.

As sequencing technologies become more affordable, it is now realistic to propose studying the evolutionary history of virtually any organism on a genomic scale. However, when dealing with non-model organisms it is not always easy to choose the best approach given a specific biological question, a limited budget, and challenging sample material. Furthermore, although recent advances in technology offer unprecedented opportunities for research in non-model organisms, they also demand unprecedented awareness from the researcher regarding the assumptions and limitations of each method. In this review we present an overview of the current sequencing technologies and the methods used in typical high-throughput data analysis pipelines. Subsequently, we contextualize high-throughput DNA sequencing technologies within their applications in non-model organism biology. We include tips regarding managing unconventional sample material, comparative and population genetic approaches that do not require fully assembled genomes, and advice on how to deal with low depth sequencing data.

The effect of presence of infected neighbouring farms for the Campylobacter infection status in Danish broiler farms.

Neighbourhood risk factors for Campylobacter infection in Danish broilers were evaluated. Campylobacter infection status of a flock was identified by PCR analysis of cloacal swab samples collected as a part of national surveillance program. Included into the study were, in total, 10,876 broiler flocks sent by 226 farms to processing plants from 2007 to 2009. A multivariable logistic regression model with autocorrelation structure was used to model the effect of exposure variables on the probability of being tested positive to Campylobacter. Results showed a significant protective effect with the absence of infected neighbours within a distance of 2 km. The analysis was adjusted for potential confounding factors. Seasonal cyclic patterns of the Campylobacter infection was accounted for by using sine and cosine function. Predicted probability maps showed a heterogeneous spatial and temporal risk of Campylobacter infection in Danish broiler.

Identification of biosecurity measures and spatial variables as potential risk factors for Aleutian disease in Danish mink farms.

Eradication of Aleutian disease was initiated in Denmark in 1976. The prevalence of positive farms has since then been reduced from 100% to only being continuously present in the region of Vendsyssel, Northern Denmark since 2004. In this study, we attempted to identify risk factors for the infection in this region based on logistic regression of spatial (environmental, neighbourhood) variables and biosecurity measures. Information on potential biosecurity (management) risk factors in the region was obtained from interviews in 342 registered farms in the region using a structured questionnaire. A total of 279 questionnaires were completed (response rate 82%). Additional spatial variables were included in the analysis. The study shows that farm size (the number of animals in the farm) and proportion of infected neighbouring farms were significant risk factors for infection with Aleutian Mink Disease Virus. These factors account for 35% of the variation of the infection status of mink farms located in Vendsyssel during 2009. These results indicate that only a coordinated effort from the farmers in the area will succeed in eradicating the disease from Denmark, because individual farms that have eradicated the disease will be at risk of re-infection from test-positive neighbours.

Molecular epidemiology of circulating highly pathogenic avian influenza (H5N1) virus in chickens, in Bangladesh, 2007-2010.

Bangladesh has been severely hit by highly pathogenic avian influenza H5N1 (HPAI-H5N1). However, little is known about the genetic diversity and the evolution of the circulating viruses in Bangladesh. In the present study, we analyzed the hemagglutinin gene of 30 Bangladeshi chicken isolates from 2007 through 2010. We analyzed the polybasic amino acid sequence motif of the cleavage site and amino acid substitution pattern. Phylogenetic history was reconstructed using neighbor-joining and Bayesian time-scaled methods. In addition, we used Mantel correlation tests to analyze the relation between genetic relatedness and spatial and temporal distances. Neighbor-joining phylogeography revealed that virus circulating in Bangladesh from 2007 through 2010 belonged to clade 2.2. The results suggest that clade 2.2 viruses are firmly entrenched and have probably become endemic in Bangladesh. We detected several amino acid substitutions, but they are not indicative of adaptation toward human infection. The Mantel correlation test confirmed significant correlation between genetic distances and temporal distances between the viruses. The Bayesian tree shows that isolates from waves 3 and 4 derived from a subgroup of isolates from the previous waves grouping with a high posterior probability (pp=1.0). This indicates the possibility of formation of local subclades. One surprising finding of spatio-temporal analysis was that genetically identical virus caused independent outbreaks over a distance of more than 200 km and within 14 days of each other. This might indicate long distance dispersal through vectors such as migratory birds and vehicles, and challenges the effectiveness of movement restriction around 10 km radius of an outbreak. The study indicates possible endemicity of the clade 2.2 HPAI-H5N1 virus in Bangladesh. Furthermore, the formation of a subclade capable of transmission to humans cannot be ruled out. The findings of this study might provide valuable information for future surveillance, prevention and control programme.

Persistent spatial clusters of plasmacytosis among Danish mink farms.

Aleutian disease (Plasmacytosis) is caused by the Aleutian mink disease virus (AMDV), an autonomous parvovirus and affects many mustelid species, including the American mink (Neovisonvison). In Denmark, an eradication program reduced the prevalence of test-positive farms from 100% in 1976 to 15% in 1996. Nevertheless, the disease persists in the Vendsyssel district of Northern Jutland, despite the eradication efforts. In this study, we used spatial epidemiological analysis to test for spatial autocorrelation of the distribution of farms positive for the disease. We investigated 2375 farms in Denmark (342 of which were located in the Vendsyssel district), during the period 2000-2008. For the purpose of our study, a farm was considered positive when, on any test conducted in a year, at least three animals were tested positive. To detect spatial clusters, we performed a retrospective analysis with spatial scan statistics. We performed one analysis for each of the nine years (2000-2008). A separate analysis was conducted with only the farms in Vendsyssel included. The spatial cluster analysis revealed a significant cluster throughout the time period studied in Northern Jutland. The only exception was 2002 when an outbreak was detected in the southern part of Jutland, and not in the north. The farm-level prevalence of the disease in Denmark was highest in this year, suggesting that the outbreak in the south could have masked the persistent signal from the north; the northern cluster was still significant when analysing only the Vendsyssel populations. These results confirm that Northern Jutland continues to have a significantly higher number of cases than expected if the disease was randomly distributed.