Scientific highlights

As the only endemic neotropical parrot to have recently lived in the northern hemisphere, the Carolina parakeet (Conuropsis carolinensis) was an iconic North American bird. The last surviving specimen died in the Cincinnati Zoo in 1918 [1]. The cause of its extinction remains contentious: besides excessive mortality associated to habitat destruction and active hunting, their survival could have been negatively affected by its range having become increasingly patchy [2] or by the exposure to poultry pathogens [3, 4]. In addition, the Carolina parakeet showed a predilection for cockleburs, an herbaceous plant that contains a powerful toxin, carboxyatractyloside, or CAT [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To explore the demographic history of this bird, we generated the complete genomic sequence of a preserved specimen held in a private collection in Espinelves (Girona, Spain), as well as of a close extant relative, Aratinga solstitialis. We identified two non-synonymous genetic changes in two highly conserved proteins known to interact with CAT that could underlie a specific dietary adaptation to this toxin. Our genomic analyses did not reveal evidence of a dramatic past demographic decline in the Carolina parakeet; also, its genome did not exhibit the long runs of homozygosity that are signals of recent inbreeding and are typically found in endangered species. As such, our results suggest its extinction was an abrupt process and thus likely solely attributable to human causes.

The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a novel library preparation method for high throughput barcoding of short reads where millions of random barcodes can be used to reconstruct megabase-scale phase blocks.

In wheatears and related species ('open-habitat chats'), molecular phylogenetics has led to a comprehensively revised understanding of species relationships and species diversity. Phylogenetic analyses have suggested that, in many cases, phenotypic similarities do not reflect species' relationships, revealing traditionally defined genera as non-monophyletic. This led to the suggestion of pervasive parallel evolution of open-habitat chats' plumage coloration and ecological phenotypes. However, to date, the molecular evidence for the phylogenetic relationships among open-habitat chats is mainly limited to mitochondrial DNA. Here, we assessed whether the mitochondrial relationships are supported by genome-wide data. To this end, we reconstructed the species tree among 14 open-habitat chat taxa using multi-species coalescent analyses based on ~1'300 SNPs. Our results confirm previous ones based chiefly on mitochondrial DNA; notably the paraphyly of the Oenanthe lugens complex and the clustering of individual species formerly placed in the genera Cercomela and Myrmecocichla within Oenanthe. Since several variable morphological and ecological characteristics occur in multiple places across the open-habitat chat phylogeny, our study consolidates the evidence for pervasive parallel evolution in the plumage coloration and ecology of open-habitat chats.

Despite considerable progress in schizophrenia genetics, most findings have been for large rare structural variants and common variants in well-imputed regions with few genes implicated from exome sequencing. Whole genome sequencing (WGS) can potentially provide a more complete enumeration of etiological genetic variation apart from the exome and regions of high linkage disequilibrium. We analyze high-coverage WGS data from 1162 Swedish schizophrenia cases and 936 ancestry-matched population controls. Our main objective is to evaluate the contribution to schizophrenia etiology from a variety of genetic variants accessible to WGS but not by previous technologies. Our results suggest that ultra-rare structural variants that affect the boundaries of topologically associated domains (TADs) increase risk for schizophrenia. Alterations in TAD boundaries may lead to dysregulation of gene expression. Future mechanistic studies will be needed to determine the precise functional effects of these variants on biology.

Population health research is increasingly focused on the genetic determinants of healthy ageing, but there is no public resource of whole genome sequences and phenotype data from healthy elderly individuals. Here we describe the first release of the Medical Genome Reference Bank (MGRB), comprising whole genome sequence and phenotype of 2570 elderly Australians depleted for cancer, cardiovascular disease, and dementia. We analyse the MGRB for single-nucleotide, indel and structural variation in the nuclear and mitochondrial genomes. MGRB individuals have fewer disease-associated common and rare germline variants, relative to both cancer cases and the gnomAD and UK Biobank cohorts, consistent with risk depletion. Age-related somatic changes are correlated with grip strength in men, suggesting blood-derived whole genomes may also provide a biologic measure of age-related functional deterioration. The MGRB provides a broadly applicable reference cohort for clinical genetics and genomic association studies, and for understanding the genetics of healthy ageing.

Genomic rearrangements associated with speciation often result in chromosome number variation among closely related species. Malassezia species show variable karyotypes ranging between 6 and 9 chromosomes. Here, we experimentally identified all 8 centromeres in M. sympodialis as 3 to 5 kb long kinetochore-bound regions spanning an AT-rich core and depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur with 7 chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with 9 chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred 9-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.

Background: We have reported that BRAF V600E mutations and microsatellite instability-high (MSI-H) are more prevalent in a population-based cohort of metastatic colorectal cancer (mCRC) patients than has been reported from clinical trials or hospital-based patient groups. The aim was to explore if other mutations in mCRC differ in prevalence between these cohorts in relation to mismatch repair status and primary tumor location and if presence of bone or brain metastases is associated with any mutations.Material and methods: A population-based cohort of 798 mCRC patients from three regions in Scandinavia was used. Forty-four cancer related genes were investigated in a custom designed Ampliseq hotspot panel. Differences in survival were analyzed using the Kaplan-Meier estimator and the Cox regression analysis.Results: Determination of mutations was possible in 449/501 patients for 40/44 genes. Besides BRAF V600E, seen in 19% of the tumors, none of the other mutations appeared more prevalent than in trial cohorts. BRAF V600E and MSI-H, seen in 8%, were associated with poor prognosis as was right-sided primary tumor location (39%) when compared to left-sided and rectum together; however, in a multivariable regression, only the BRAF mutation retained its statistical significance. No other mutations were associated with poor prognosis. ERBB2 alterations were more common if bone metastases were present at diagnosis (17% vs. 4%, p = .011). No association was found for brain metastases. Fifty-two percent had an alteration that is treatable with an FDA-approved targeted therapy, chiefly by EGFR-inhibitor for RAS wild-type and a check-point inhibitor for MSI-H tumors.Conclusions: Right-sided tumor location, BRAF V600E mutations, but no other investigated mutation, and MSI-H are more commonly seen in an unselected cohort than is reported from clinical patient cohorts, likely because they indicate poor prognosis. Half of the patients have a tumor that is treatable with an already FDA-approved targeted drug for mCRC.

There is a need to accurately call human leukocyte antigen (HLA) genes from existing short-read sequencing data, however there is no single solution that matches the gold standard of Sanger sequenced lab typing. Here we aimed to combine results from available software programs, minimizing the biases of applied algorithm and HLA reference. The result is a robust HLA population resource for the published 1000 Swedish genomes, and a framework for future HLA interrogation. HLA 2nd-field alleles were called using four imputation and inference methods for the classical eight genes (class I: HLA-A, HLA-B, HLA-C; class II: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1). A high confidence population set (SweHLA) was determined using an n-1 concordance rule for class I (four software) and class II (three software) alleles. Results were compared across populations and individual programs benchmarked to SweHLA. Per gene, 875 to 988 of the 1000 samples were genotyped in SweHLA; 920 samples had at least seven loci called. While a small fraction of reference alleles were common to all software (class I = 1.9% and class II = 4.1%), this did not affect the overall call rate. Gene-level concordance was high compared to European populations (>0.83%), with COX and PGF the dominant SweHLA haplotypes. We noted that 15/18 discordant alleles (delta allele frequency >2) were previously reported as disease-associated. These differences could in part explain across-study genetic replication failures, reinforcing the need to use multiple software solutions. SweHLA demonstrates a way to use existing NGS data to generate a population resource agnostic to individual HLA software biases.

Genes with sex-biased expression show a number of unique properties and this has been seen as evidence for conflicting selection pressures in males and females, forming a genetic 'tug-of-war' between the sexes. However, we lack studies of taxa where an understanding of conflicting phenotypic selection in the sexes has been linked with studies of genomic signatures of sexual conflict. Here, we provide such a link. We used an insect where sexual conflict is unusually well understood, the seed beetle Callosobruchus maculatus, to test for molecular genetic signals of sexual conflict across genes with varying degrees of sex-bias in expression. We sequenced, assembled and annotated its genome and performed population resequencing of three divergent populations. Sex-biased genes showed increased levels of genetic diversity and bore a remarkably clear footprint of relaxed purifying selection. Yet, segregating genetic variation was also affected by balancing selection in weakly female-biased genes, while male-biased genes showed signs of overall purifying selection. Female-biased genes contributed disproportionally to shared polymorphism across populations, while male-biased genes, male seminal fluid protein genes and sex-linked genes did not. Genes showing genomic signatures consistent with sexual conflict generally matched life-history phenotypes known to experience sexually antagonistic selection in this species. Our results highlight metabolic and reproductive processes, confirming the key role of general life-history traits in sexual conflict.

Plasmodiophora brassicae is a soil-borne pathogen that attacks roots of cruciferous plants causing clubroot disease. The pathogen belongs to the Plasmodiophorida order in Phytomyxea. Here we used long-read SMRT technology to clarify the P. brassicae e3 genomic constituents along with comparative and phylogenetic analyses. Twenty contigs representing the nuclear genome and one mitochondrial (mt) contig were generated, together comprising 25.1 Mbp. Thirteen of the 20 nuclear contigs represented chromosomes from telomere to telomere characterized by [TTTTAGGG] sequences. Seven active gene candidates encoding synaptonemal complex-associated and meiotic-related protein homologs were identified, a finding that argues for possible genetic recombination events. The circular mt genome is large (114,663 bp), gene dense and intron rich. It shares high synteny with the mt genome of Spongospora subterranea, except in a unique 12 kb region delimited by shifts in GC content and containing tandem minisatellite- and microsatellite repeats with partially palindromic sequences. De novo annotation identified 32 protein-coding genes, 28 structural RNA genes and 19 ORFs. ORFs predicted in the repeat-rich region showed similarities to diverse organisms suggesting possible evolutionary connections. The data generated here form a refined platform for the next step involving functional analysis, all to clarify the complex biology of P. brassicae.

Future climate change has been predicted to disrupt local adaptation in many perennial plants, such as forest trees, but the magnitude and location of these effects are thus far poorly understood. Here, we assess local adaptation to current climate in European aspen ( Populus tremula) by using environmental association analyses to identify genetic variants associated with two representative climate variables describing current day variation in temperature and precipitation. We also analysed patterns of genetic differentiation between southern and northern populations and observe that regions of high genetic differentiation are enriched for SNPs that are significantly associated with climate. Using variants associated with climate, we examined patterns of isolation by distance and environment and used spatial modelling to predict the geographic distribution of genomic variation in response to two scenarios of future climate change. We show that climate conditions at a northern reference site will correspond to climate conditions experienced by current day populations located 4-8 latitude degrees further south. By assessing the relationship between phenotypic traits and vegetative fitness, we also demonstrate that southern populations harbour genetic variation that likely would be adaptive further north under both climate change scenarios. Current day populations at the lagging edge of the distribution in Sweden can therefore serve as sources for introducing adaptive alleles onto northern populations, but the likelihood of this largely depends on naturally occurring levels of gene flow.

Characterizing the relationship between genetic, epigenetic (e.g., deoxyribonucleic acid [DNA] methylation), and transcript variation could provide insights into mechanisms regulating hemostasis and potentially identify new drug targets. Several hemostatic factors are synthesized in the liver, yet high-resolution DNA methylation data from human liver tissue is currently lacking for these genes. Single-nucleotide polymorphisms (SNPs) can influence DNA methylation in cis which can affect gene expression. This can be analyzed through allele-specific methylation (ASM) experiments. We performed targeted genomic DNA- and bisulfite-sequencing of 35 hemostatic genes in human liver samples for SNP and DNA methylation analysis, respectively, and integrated the data for ASM determination. ASM-associated SNPs (ASM-SNPs) were tested for association to gene expression in liver using in-house generated ribonucleic acid-sequencing data. We then assessed whether ASM-SNPs associated with gene expression, plasma proteins, or other traits relevant for hemostasis using publicly available data. We identified 112 candidate ASM-SNPs. Of these, 68% were associated with expression of their respective genes in human liver or in other human tissues and 54% were associated with the respective plasma protein levels, activity, or other relevant hemostatic genome-wide association study traits such as venous thromboembolism, coronary artery disease, stroke, and warfarin dose maintenance. Our study provides the first detailed map of the DNA methylation landscape and ASM analysis of hemostatic genes in human liver tissue, and suggests that methylation regulated by genetic variants in cis may provide a mechanistic link between noncoding SNPs and variation observed in circulating hemostatic proteins, prothrombotic diseases, and drug response.

Ancient DNA provides a powerful means to investigate the timing, rate and extent of population declines caused by extrinsic factors, such as past climate change and human activities. One species probably affected by both these factors is the arctic fox, which had a large distribution during the last glaciation that subsequently contracted at the start of the Holocene. More recently, the arctic fox population in Scandinavia went through a demographic bottleneck owing to human persecution. To investigate the consequences of these processes, we generated mitogenome sequences from a temporal dataset comprising Pleistocene, historical and modern arctic fox samples. We found no evidence that Pleistocene populations in mid-latitude Europe or Russia contributed to the present-day gene pool of the Scandinavian population, suggesting that postglacial climate warming led to local population extinctions. Furthermore, during the twentieth-century bottleneck in Scandinavia, at least half of the mitogenome haplotypes were lost, consistent with a 20-fold reduction in female effective population size. In conclusion, these results suggest that the arctic fox in mainland Western Europe has lost genetic diversity as a result of both past climate change and human persecution. Consequently, it might be particularly vulnerable to the future challenges posed by climate change. This article is part of a discussion meeting issue 'The past is a foreign country: how much can the fossil record actually inform conservation?'

Blood is the predominant source for molecular analyses in humans, both in clinical and research settings. It is the target for many therapeutic strategies, emphasizing the need for comprehensive molecular maps of the cells constituting human blood. In this study, we performed a genome-wide transcriptomic analysis of protein-coding genes in sorted blood immune cell populations to characterize the expression levels of each individual gene across the blood cell types. All data are presented in an interactive, open-access Blood Atlas as part of the Human Protein Atlas and are integrated with expression profiles across all major tissues to provide spatial classification of all protein-coding genes. This allows for a genome-wide exploration of the expression profiles across human immune cell populations and all major human tissues and organs.

Introduction. Cervical cancer is caused by human papillomavirus (HPV), but some cases may test HPV-negative. We previously tested 2850 Swedish cases and found that 394/2850 (13.8 %) cases tested HPV DNA-negative by PCR. Sequencing is the most thorough method to assess HPV status.Aim.. We wished to assess whether deep sequencing might detect HPV sequences among these HPV-negative cervical cancer specimens, and to increase the likelihood of detecting transcriptionally active infections.Methodology. Out of the 2850 cancer cases, we sequenced a random sample of 92 HPV PCR-negative cervical cancers and 34 HPV PCR-positive cervical cancers. Four pools of blank blocks were sequenced as negative controls. To enrich for mRNA - a hallmark of active viral infection - the samples were extracted, reverse-transcribed, rRNA-depleted and then sequenced using the NovaSeq 6000 system (Illumina, USA). High-quality reads were aligned to the human genome and non-human reads were queried against HPV proteins.Results. We obtained a median of 23 million paired reads per sample. HPV was detected in 31/34 HPV PCR-positive cases. Among cases negative for HPV by PCR, 48/92 (52.2 %) contained HPV sequences, with HPV33 being the most commonly detected type among these (14/48 cases, 29.2 %). Comparison of the ratio of exon and intron sequences found that the sequenced material contained both DNA and RNA. Splice junctions were detected in 12 cases.Conclusion. Apparently, some cervical cancers contain HPV that is difficult to detect by PCR. Sequencing may be a helpful tool for additional quality assurance for HPV testing methods.

Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: either yellow/orange or white. Here we map the locus associated with this ALHS in Colias crocea to a transposable element insertion located downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find white-specific expression of BarH-1 suppresses the formation of pigment granules in wing scales and gives rise to white wing color. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. Together, these findings characterize a mechanism for a female-limited ALHS.

Craniosynostosis (CS), the premature closure of one or more cranial sutures, occurs both as part of a syndrome or in isolation (nonsyndromic form). Here, we have studied the prevalence and spectrum of genetic alterations associated with coronal suture closure in 100 Scandinavian patients treated at a single craniofacial unit. All patients were phenotypically assessed and analyzed with a custom-designed 63 gene NGS-panel. Most cases (78%) were syndromic forms of CS. Pathogenic and likely pathogenic variants explaining the phenotype were found in 80% of the families with syndromic CS and in 14% of those with nonsyndromic CS. Sixty-five percent of the families had mutations in the CS core genes FGFR2, TWIST1, FGFR3, TCF12, EFNB1, FGFR1, and POR. Five novel pathogenic/likely pathogenic variants in TWIST1, TCF12, and EFNB1 were identified. We also found novel variants in SPECC1L, IGF1R, and CYP26B1 with a possible modulator phenotypic effect. Our findings demonstrate that NGS targeted sequencing is a powerful tool to detect pathogenic mutations in patients with coronal CS and further emphasize the importance of thorough assessment of the patient's phenotype for reliable interpretation of the molecular findings. This is particularly important in patients with complex phenotypes and rare forms of CS.

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein-Elba3-bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

One of the most complex organs in the human body is the testis, where spermatogenesis takes place. This physiological process involves thousands of genes and proteins that are activated and repressed, making testis the organ with the highest number of tissue-specific genes. However, the function of a large proportion of the corresponding proteins remains unknown and testis harbors many missing proteins (MPs), defined as products of protein-coding genes that lack experimental mass spectrometry evidence. Here, an integrated omics approach was used for exploring the cell type-specific protein expression of genes with an elevated expression in testis. By combining genome-wide transcriptomics analysis with immunohistochemistry, more than 500 proteins with distinct testicular protein expression patterns were identified, and these were selected for in-depth characterization of their in situ expression in eight different testicular cell types. The cell type-specific protein expression patterns allowed us to identify six distinct clusters of expression at different stages of spermatogenesis. The analysis highlighted numerous poorly characterized proteins in each of these clusters whose expression overlapped with that of known proteins involved in spermatogenesis, including 85 proteins with an unknown function and 60 proteins that previously have been classified as MPs. Furthermore, we were able to characterize the in situ distribution of several proteins that previously lacked spatial information and cell type-specific expression within the testis. The testis elevated expression levels both at the RNA and protein levels suggest that these proteins are related to testis-specific functions. In summary, the study demonstrates the power of combining genome-wide transcriptomics analysis with antibody-based protein profiling to explore the cell type-specific expression of both well-known proteins and MPs. The analyzed proteins constitute important targets for further testis-specific research in male reproductive disorders.