National Bioinformatics Infrastructure Sweden

Reptiles use pterin and carotenoid pigments to produce yellow, orange, and red colors. These conspicuous colors serve a diversity of signaling functions, but their molecular basis remains unresolved. Here, we show that the genomes of sympatric color morphs of the European common wall lizard ( Podarcis muralis ), which differ in orange and yellow pigmentation and in their ecology and behavior, are virtually undifferentiated. Genetic differences are restricted to two small regulatory regions near genes associated with pterin [ sepiapterin reductase ( SPR )] and carotenoid [ beta-carotene oxygenase 2 ( BCO2 )] metabolism, demonstrating that a core gene in the housekeeping pathway of pterin biosynthesis has been coopted for bright coloration in reptiles and indicating that these loci exert pleiotropic effects on other aspects of physiology. Pigmentation differences are explained by extremely divergent alleles, and haplotype analysis revealed abundant transspecific allele sharing with other lacertids exhibiting color polymorphisms. The evolution of these conspicuous color ornaments is the result of ancient genetic variation and cross-species hybridization.

Tumor-associated macrophages (TAMs) are attractive targets for immunotherapy. Recently, studies in animal models showed that treatment with an anti-TAM antibody directed against the scavenger receptor MARCO resulted in suppression of tumor growth and metastatic dissemination. Here we investigated the expression of MARCO in relation to other macrophage markers and immune pathways in a non-small cell lung cancer (NSCLC) cohort (n = 352). MARCO, CD68, CD163, MSR1 and programmed death ligand-1 (PD-L1) were analyzed by immunohistochemistry and immunofluorescence, and associations to other immune cells and regulatory pathways were studied in a subset of cases (n = 199) with available RNA-seq data. We observed a large variation in macrophage density between cases and a strong correlation between CD68 and CD163, suggesting that the majority of TAMs present in NSCLC exhibit a protumor phenotype. Correlation to clinical data only showed a weak trend toward worse survival for patients with high macrophage infiltration. Interestingly, MARCO was expressed on a distinct subpopulation of TAMs, which tended to aggregate in close proximity to tumor cell nests. On the transcriptomic level, we found a positive association between MARCO gene expression and general immune response pathways including strong links to immunosuppressive TAMs, T-cell infiltration and immune checkpoint molecules. Indeed, a higher macrophage infiltration was seen in tumors expressing PD-L1, and macrophages residing within tumor cell nests co-expressed MARCO and PD-L1. Thus, MARCO is a potential new immune target for anti-TAM treatment in a subset of NSCLC patients, possibly in combination with available immune checkpoint inhibitors.

// Magnus Tobiasson 1, * , Hani Abdulkadir 1, * , Andreas Lennartsson 2 , Shintaro Katayama 2 , Francesco Marabita 3, 4 , Ayla De Paepe 1 , Mohsen Karimi 1 , Kaarel Krjutskov 2, 5, 6 , Elisabet Einarsdottir 2, 5 , Michael Grövdal 1 , Monika Jansson 1 , Asmaa Ben Azenkoud 1 , Lina Corddedu 2 , Sören Lehmann 1, 7 , Karl Ekwall 2 , Juha Kere 2, 5 , Eva Hellström-Lindberg 1, * and Johanna Ungerstedt 1, * 1 Center for Hematology and Regenerative Medicine, Department of Medicine Huddinge, Division of Hematology Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden 2 Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden 3 Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden 4 National Bioinformatics Infrastructure Sweden, Stockholm, Sweden 5 Molecular Neurology Research Program, University of Helsinki, and Folkhälsan Institute of Genetics, Helsinki, Finland 6 Competence Centre on Health Technologies, Tartu, Estonia 7 Department of Medical Sciences, Uppsala University, Uppsala, Sweden * These authors have contributed equally to this work Correspondence to: Magnus Tobiasson, email: johanna.ungerstedt@ki.se Keywords: MDS, azacitidine, epigenetics, DNA methylation, histone modifications Received: November 09, 2016     Accepted: February 01, 2017     Published: February 28, 2017 ABSTRACT Azacitidine (Aza) is first-line treatment for patients with high-risk myelodysplastic syndromes (MDS), although its precise mechanism of action is unknown. We performed the first study to globally evaluate the epigenetic effects of Aza on MDS bone marrow progenitor cells assessing gene expression (RNA seq), DNA methylation (Illumina 450k) and the histone modifications H3K18ac and H3K9me3 (ChIP seq). Aza induced a general increase in gene expression with 924 significantly upregulated genes but this increase showed no correlation with changes in DNA methylation or H3K18ac, and only a weak association with changes in H3K9me3. Interestingly, we observed activation of transcripts containing 15 endogenous retroviruses (ERVs) confirming previous cell line studies. DNA methylation decreased moderately in 99% of all genes, with a median β-value reduction of 0.018; the most pronounced effects seen in heterochromatin. Aza-induced hypomethylation correlated significantly with change in H3K9me3. The pattern of H3K18ac and H3K9me3 displayed large differences between patients and healthy controls without any consistent pattern induced by Aza. We conclude that the marked induction of gene expression only partly could be explained by epigenetic changes, and propose that activation of ERVs may contribute to the clinical effects of Aza in MDS.

A genomic database of all Earth's eukaryotic species could contribute to many scientific discoveries; however, only a tiny fraction of species have genomic information available. In 2018, scientists across the world united under the Earth BioGenome Project (EBP), aiming to produce a database of high-quality reference genomes containing all ~1.5 million recognized eukaryotic species. As the European node of the EBP, the European Reference Genome Atlas (ERGA) sought to implement a new decentralised, equitable and inclusive model for producing reference genomes. For this, ERGA launched a Pilot Project establishing the first distributed reference genome production infrastructure and testing it on 98 eukaryotic species from 33 European countries. Here we outline the infrastructure and explore its effectiveness for scaling high-quality reference genome production, whilst considering equity and inclusion. The outcomes and lessons learned provide a solid foundation for ERGA while offering key learnings to other transnational, national genomic resource projects and the EBP.

OBJECTIVE: The state-of-the-art genome annotation tools output GFF3 format files, while this format is not accepted as submission format by the International Nucleotide Sequence Database Collaboration (INSDC) databases. Converting the GFF3 format to a format accepted by one of the three INSDC databases is a key step in the achievement of genome annotation projects. However, the flexibility existing in the GFF3 format makes this conversion task difficult to perform. Until now, no converter is able to handle any GFF3 flavour regardless of source. RESULTS: Here we present EMBLmyGFF3, a robust universal converter from GFF3 format to EMBL format compatible with genome annotation submission to the European Nucleotide Archive. The tool uses json parameter files, which can be easily tuned by the user, allowing the mapping of corresponding vocabulary between the GFF3 format and the EMBL format. We demonstrate the conversion of GFF3 annotation files from four different commonly used annotation tools: Maker, Prokka, Augustus and Eugene. EMBLmyGFF3 is freely available at https://github.com/NBISweden/EMBLmyGFF3 .

mediate many important chemical transformations in the biosphere from degrading abundant polymers to synthesis of toxic compounds. Two of the most pressing issues in microbial interactions are how consortia are established and how we can modulate these microbial communities to express desirable functions. Here, we propose that public goods (i.e., metabolites of high energy demand in biosynthesis) facilitate energy conservation for life under energy-limited conditions and determine the assembly and function of the consortia. Our report suggests that an understanding of public good dynamics could result in new ways to improve microbial pollutant degradation in anaerobic systems.

The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides β-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and β-catenin, leading to elevated luciferase reporter of β-catenin/Lef1-mediated transcriptional activation. The mechanism for the reduced β-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of β-catenin/Lef1-mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells.

Domestication has resulted in immense phenotypic changes in animals despite their relatively short evolutionary history. The European rabbit is one of the most recently domesticated animals, but exhibits distinct morphological, physiological, and behavioral differences from their wild conspecifics. A previous study revealed that sequence variants with striking allele frequency differences between wild and domestic rabbits were enriched in conserved noncoding regions, in the vicinity of genes involved in nervous system development. This suggests that a large proportion of the genetic changes targeted by selection during domestication might affect gene regulation. Here, we generated RNA-sequencing data for four brain regions (amygdala, hypothalamus, hippocampus, and parietal/temporal cortex) sampled at birth and revealed hundreds of differentially expressed genes (DEGs) between wild and domestic rabbits. DEGs in amygdala were significantly enriched for genes associated with dopaminergic function and all 12 DEGs in this category showed higher expression in domestic rabbits. DEGs in hippocampus were enriched for genes associated with ciliary function, all 21 genes in this category showed lower expression in domestic rabbits. These results indicate an important role of dopamine signaling and ciliary function in the evolution of tameness during rabbit domestication. Our study shows that gene expression in specific pathways has been profoundly altered during domestication, but that the majority of genes showing differential expression in this study have not been the direct targets of selection.

This study examines evolutionary and ecological relationships of three of the most ubiquitous and abundant freshwater bacterial genera: “ Ca . Planktophila” (acI-A), “ Ca. Nanopelagicus” (acI-B), and “ Ca. Fonsibacter” (LD12). Due to high abundance, these genera might have a significant influence on nutrient cycling in freshwaters worldwide, and this study adds a layer of understanding to how seemingly competing clades of bacteria can coexist by having different cooperation strategies. Our synthesis ties together network and ecological theory with empirical evidence and lays out a framework for how the functioning of populations within complex microbial communities can be studied.

Species invasion and range expansion are currently under scrutiny due to increasing anthropogenic impact on the natural environment. This is also true for harmful algal blooms, which have been reported to have increased in frequency. However, this research is challenging due to the ephemeral nature, small size and mostly low concentrations of microalgae in the environment. One such species is the nuisance microalga Gonyostomum semen (Raphidophyceae), which has increased in occurrence in northern Europe in recent decades. The question of whether the species has expanded its habitat range or if it was already present in the lakes but was too rare to be detected remains unanswered. The aim of the present study was to determine the genetic structure and dispersal pathways of G. semen using RAD (restriction-site-associated DNA) tag sequencing. For G. semen, which has a huge genome (32 Gbp), we faced particular challenges, but were nevertheless able to recover over 1000 single nucleotide polymorphisms at high coverage. Our data revealed a distinct population genetic structure, demonstrating a divide of western and eastern populations that probably represent different lineages. Despite significant genetic differentiation among lakes, we found only limited isolation-by-distance. While we had expected a pattern of recent expansion northwards, the data demonstrated gene flow from the northeast/east towards the southwest/west. This genetic signature suggests that the observed gene flow may be due to dispersal by autumn migratory birds, which act as dispersal vectors of resistant resting propagules that form at the end of the G. semen blooms.

The escape of DNA from mitochondria into the nuclear genome (nuclear mitochondrial DNA, NUMT) is an ongoing process. Although pervasively observed in eukaryotic genomes, their evolutionary trajectories in a mammal-wide context are poorly understood. The main challenge lies in the orthology assignment of NUMTs across species due to their fast evolution and chromosomal rearrangements over the past 200 million years. To address this issue, we systematically investigated the characteristics of NUMT insertions in 45 mammalian genomes and established a novel, synteny-based method to accurately predict orthologous NUMTs and ascertain their evolution across mammals. With a series of comparative analyses across taxa, we revealed that NUMTs may originate from nonrandom regions in mtDNA, are likely found in transposon-rich and intergenic regions, and unlikely code for functional proteins. Using our synteny-based approach, we leveraged 630 pairwise comparisons of genome-wide microsynteny and predicted the NUMT orthology relationships across 36 mammals. With the phylogenetic patterns of NUMT presence-and-absence across taxa, we constructed the ancestral state of NUMTs given the mammal tree using a coalescent method. We found support on the ancestral node of Fereuungulata within Laurasiatheria, whose subordinal relationships are still controversial. This study broadens our knowledge on NUMT insertion and evolution in mammalian genomes and highlights the merit of NUMTs as alternative genetic markers in phylogenetic inference.

A global genome database of all of Earth's species diversity could be a treasure trove of scientific discoveries. However, regardless of the major advances in genome sequencing technologies, only a tiny fraction of species have genomic information available. To contribute to a more complete planetary genomic database, scientists and institutions across the world have united under the Earth BioGenome Project (EBP), which plans to sequence and assemble high-quality reference genomes for all ~1.5 million recognized eukaryotic species through a stepwise phased approach. As the initiative transitions into Phase II, where 150,000 species are to be sequenced in just four years, worldwide participation in the project will be fundamental to success. As the European node of the EBP, the European Reference Genome Atlas (ERGA) seeks to implement a new decentralised, accessible, equitable and inclusive model for producing high-quality reference genomes, which will inform EBP as it scales. To embark on this mission, ERGA launched a Pilot Project to establish a network across Europe to develop and test the first infrastructure of its kind for the coordinated and distributed reference genome production on 98 European eukaryotic species from sample providers across 34 European countries. Here we outline the process and challenges faced during the development of a pilot infrastructure for the production of reference genome resources, and explore the effectiveness of this approach in terms of high-quality reference genome production, considering also equity and inclusion. The outcomes and lessons learned during this pilot provide a solid foundation for ERGA while offering key learnings to other transnational and national genomic resource projects.

Data Management Plans are now considered a key element of Open Science. They describe the data management life cycle for the data to be collected, processed and/or generated within the lifetime of a particular project or activity. A Software Manag ement Plan (SMP) plays the same role but for software. Beyond its management perspective, the main advantage of an SMP is that it both provides clear context to the software that is being developed and raises awareness. Although there are a few SMPs already available, most of them require significant technical knowledge to be effectively used. ELIXIR has developed a low-barrier SMP, specifically tailored for life science researchers, aligned to the FAIR Research Software principles. Starting from the Four Recommendations for Open Source Software, the ELIXIR SMP was iteratively refined by surveying the practices of the community and incorporating the received feedback. Currently available as a survey, future plans of the ELIXIR SMP include a human- and machine-readable version, that can be automatically queried and connected to relevant tools and metrics within the ELIXIR Tools ecosystem and beyond.

ELIXIR is a pan-European intergovernmental organisation for life science that aims to coordinate bioinformatics resources in a single infrastructure across Europe; bioinformatics training is central to its strategy, which aims to develop a training community that spans all ELIXIR member states. In an evidence-based approach for strengthening bioinformatics training programmes across Europe, the ELIXIR Training Platform, led by the ELIXIR EXCELERATE Quality and Impact Assessment Subtask in collaboration with the ELIXIR Training Coordinators Group, has implemented an assessment strategy to measure quality and impact of its entire training portfolio. Here, we present ELIXIR's framework for assessing training quality and impact, which includes the following: specifying assessment aims, determining what data to collect in order to address these aims, and our strategy for centralised data collection to allow for ELIXIR-wide analyses. In addition, we present an overview of the ELIXIR training data collected over the past 4 years. We highlight the importance of a coordinated and consistent data collection approach and the relevance of defining specific metrics and answer scales for consortium-wide analyses as well as for comparison of data across iterations of the same course.

BACKGROUND: Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) copy number alterations and unbalanced gene recombination events have been reported to occur in breast cancer. Importantly, LRIG1 loss was recently shown to predict early and late relapse in stage I-II breast cancer. METHODS: We developed droplet digital PCR (ddPCR) assays for the determination of relative LRIG1 copy numbers and used these assays to analyze LRIG1 in twelve healthy individuals, 34 breast tumor samples previously analyzed by fluorescence in situ hybridization (FISH), and 423 breast tumor cytosols. RESULTS: Four of the LRIG1/reference gene assays were found to be precise and robust, showing copy number ratios close to 1 (mean, 0.984; standard deviation, +/- 0.031) among the healthy control population. The correlation between the ddPCR assays and previous FISH results was low, possibly because of the different normalization strategies used. One in 34 breast tumors (2.9%) showed an unbalanced LRIG1 recombination event. LRIG1 copy number ratios were associated with the breast cancer subtype, steroid receptor status, ERBB2 status, tumor grade, and nodal status. Both LRIG1 loss and gain were associated with unfavorable metastasis-free survival; however, they did not remain significant prognostic factors after adjustment for common risk factors in the Cox regression analysis. Furthermore, LRIG1 loss was not significantly associated with survival in stage I and II cases. CONCLUSIONS: Although LRIG1 gene aberrations may be important determinants of breast cancer biology, and prognostic markers, the results of this study do not verify an important role for LRIG1 copy number analyses in predicting the risk of relapse in early-stage breast cancer.

Here, we present the genome of the industrial ethanol production strain Brettanomyces bruxellensis CBS 11270. The nuclear genome was found to be diploid, containing four chromosomes with sizes of ranging from 2.2 to 4.0 Mbp. A 75 Kbp mitochondrial genome was also identified. Comparing the homologous chromosomes, we detected that 0.32% of nucleotides were polymorphic, i.e. formed single nucleotide polymorphisms (SNPs), 40.6% of them were found in coding regions (i.e. 0.13% of all nucleotides formed SNPs and were in coding regions). In addition, 8,538 indels were found. The total number of protein coding genes was 4897, of them, 4,284 were annotated on chromosomes; and the mitochondrial genome contained 18 protein coding genes. Additionally, 595 genes, which were annotated, were on contigs not associated with chromosomes. A number of genes was duplicated, most of them as tandem repeats, including a six-gene cluster located on chromosome 3. There were also examples of interchromosomal gene duplications, including a duplication of a six-gene cluster, which was found on both chromosomes 1 and 4. Gene copy number analysis suggested loss of heterozygosity for 372 genes. This may reflect adaptation to relatively harsh but constant conditions of continuous fermentation. Analysis of gene topology showed that most of these losses occurred in clusters of more than one gene, the largest cluster comprising 33 genes. Comparative analysis against the wine isolate CBS 2499 revealed 88,534 SNPs and 8,133 indels. Moreover, when the scaffolds of the CBS 2499 genome assembly were aligned against the chromosomes of CBS 11270, many of them aligned completely, some have chunks aligned to different chromosomes, and some were in fact rearranged. Our findings indicate a highly dynamic genome within the species B. bruxellensis and a tendency towards reduction of gene number in long-term continuous cultivation.

Abstract An increased frequency of droughts due to anthropogenic climate change can lead to considerable stress for soil microorganisms and their functioning within northern peatlands. A better understanding of the diversity and relative abundance of methane producing and oxidizing taxa, and their functional genes, can help predict the functional potential of peatlands and how the microorganisms respond to disturbances such as drought. To address knowledge gaps in the understanding of how functional genetic diversity shifts under drought conditions, we investigated a hemi boreal mire in Southern Sweden. Environmental parameters, including soil and air temperature, precipitation, and water table depth, as well as methane flux data were collected during the summer of 2017 under typical growing conditions, and in 2018 during a drought. In addition, the diversity and composition of genes encoding for methane metabolism were determined using the captured metagenomics technique. During drought we observed a substantial increase in air and soil temperature, reduced precipitation, and a lower water table depth. Taxonomic and functional gene composition significantly changed during the drought, while diversity indices, such as alpha and beta diversity, remained similar. These results indicate that methane producing and oxidizing microbial communities, and their functional genes, displayed a resilience to drought with specific genera having the ability to outcompete others under stress. Furthermore, our results show that although methane emissions are substantially reduced during drought, we can expect to see a shift toward more resilient methanogens and methanotrophs under future climate conditions.

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

The majority of mitochondrial proteins are encoded in the nucleus, translated on cytosolic ribosomes, and subsequently targeted to the mitochondrial surface. Their further import into the organelle is facilitated by highly specialized protein translocases. Mitochondrial precursor proteins that are destined to the mitochondrial matrix and, to some extent, the inner membrane, utilize translocase of the inner membrane (TIM23). This indispensable import machinery has been extensively studied in yeast. The translocating unit of the TIM23 complex in yeast consists of two membrane proteins, Tim17 and Tim23. In contrast to previous findings, recent reports demonstrate the primary role of Tim17, rather than Tim23, in the translocation of newly synthesized proteins. Very little is known about human TIM23 translocase. Human cells have two orthologs of yeast Tim17, TIMM17A and TIMM17B. Here, using computational tools, we present the architecture of human core TIM23 variants with either TIMM17A or TIMM17B, forming two populations of highly similar complexes. The structures reveal high conservation of the core TIM23 complex between human and yeast. Interestingly, both TIMM17A and TIMM17B variants interact with TIMM23 and reactive oxygen species modulator 1 (ROMO1); a homolog of yeast Mgr2, a protein that can create a channel-like structure with Tim17. The high structural conservation of proteins that form the core TIM23 complex in yeast and humans raises an interesting question about mechanistic and functional differences that justify existence of the two variants of TIM23 in higher eukaryotes.

The formyltetrahydrofolate synthetase (FTHFS) gene is a molecular marker of choice to study the diversity of acetogenic communities. However, current analyses are limited due to lack of a high-throughput sequencing approach for FTHFS gene amplicons and a dedicated bioinformatics pipeline for data analysis, including taxonomic annotation and visualization of the sequence data. In the present study, we combined the barcode approach for multiplexed sequencing with unsupervised data analysis to visualize acetogenic community structure. We used samples from a biogas digester to develop proof-of-principle for our combined approach. We successfully generated high-throughput sequence data for the partial FTHFS gene and performed unsupervised data analysis using the novel bioinformatics pipeline "AcetoScan" presented in this study, which resulted in taxonomically annotated OTUs, phylogenetic tree, abundance plots and diversity indices. The results demonstrated that high-throughput sequencing can be used to sequence the FTHFS amplicons from a pool of samples, while the analysis pipeline AcetoScan can be reliably used to process the raw sequence data and visualize acetogenic community structure. The method and analysis pipeline described in this paper can assist in the identification and quantification of known or potentially new acetogens. The AcetoScan pipeline is freely available at https://github.com/abhijeetsingh1704/AcetoScan.