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Phylodynamic assessment of intervention strategies for the West African Ebola virus outbreak

This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (http://dx.doi.org/10.24072/pci.evolbiol.100046). The recent Ebola virus (EBOV) outbreak in West Africa witnessed considerable efforts to obtain viral genomic data as the epidemic was unfolding. If such data can be deployed in real-time, molecular epidemiological investigations could play a role in complementing contact tracing undertaken by public health agencies. Analysing the EBOV genomes accumulated to date can also deliver insights into epidemic dynamics. Such analyses have been shown that metapopulation dynamics were critical for EBOV dispersal between rural and urban areas during the epidemic, but the implications for specific intervention scenarios remain unclear. Here, we address this issue using a collection of phylodynamic approaches. We show that long-distance dispersal events (between administrative areas >250 km apart) were not crucial for epidemic expansion and that preventing viral lineage movement to any given administrative area would, in most cases, have had little impact. However, urban areas - specifically those encompassing the three capital cities and their suburbs - were critical in attracting and further disseminating the virus: preventing viral lineage movement to all three simultaneously would have contained epidemic size by two-thirds. Using continuous phylogeographic reconstructions we estimate a distance kernel for EBOV spread and reveal considerable heterogeneity in dispersal velocity through time. We also show that announcements of border closures were followed by a significant but transient effect on international virus dispersal. By quantifying the hypothetical impact of different intervention strategies as well as the impact of barriers on dispersal frequency, our study illustrates how phylodynamic analyses can help to address specific epidemiological and outbreak control questions.

epidemiology↗

Error-prone bypass of DNA lesions during lagging strand replication is a common source of germline and cancer mutations

Spontaneously occurring mutations are of great relevance in diverse fields including biochemistry, oncology, evolutionary biology, and human genetics. Studies in experimental systems have identified a multitude of mutational mechanisms including DNA replication infidelity as well as many forms of DNA damage followed by inefficient repair or replicative bypass. However, the relative contributions of these mechanisms to human germline mutations remain completely unknown. Here, based on the mutational asymmetry with respect to the direction of replication and transcription, we suggest that error-prone damage bypass on the lagging strand plays a major role in human mutagenesis. Asymmetry with respect to transcription is believed to be mediated by the action of transcription-coupled DNA repair (TC-NER). TC-NER selectively repairs DNA lesions on the transcribed strand; as a result, lesions on the non-transcribed strand are preferentially converted into mutations. In human polymorphism we detect a striking similarity between transcriptional asymmetry and asymmetry with respect to replication fork direction. This parallels the observation that damage-induced mutations in human cancers accumulate asymmetrically with respect to the direction of replication, suggesting that DNA lesions are asymmetrically resolved during replication. Re-analysis of XR-seq data, Damage-seq data and cancers with defective NER corroborate the preferential error-prone bypass of DNA lesions on the lagging strand. We experimentally demonstrate that replication delay greatly attenuates the mutagenic effect of UV-irradiation, in line with the key role of replication in conversion of DNA damage to mutations. We conservatively estimate that at least 10% of human germline mutations arise due to DNA damage rather than replication infidelity. The number of these damage-induced mutations is expected to scale with the number of replications and, consequently, paternal age.

biochemistry↗

Vulnerability correlates with life history traits and the load of deleterious mutations in fish.

Understanding why some species accumulate more deleterious substitutions than others is an important question relevant in evolutionary biology and conservation sciences. Previous studies conducted in terrestrial taxa suggest that life history traits correlate with the efficiency of purifying selection and accumulation of deleterious mutations. Using a large genome dataset of 76 species of fishes, we show that the rate of deleterious mutation accumulation (measured via dN/dS, i.e. non-synonymous over synonymous substitution rate) is associated to the vulnerability, the life-history strategies, and the latitude of species. Our results, focusing on a large clade of aquatic species, generalizes previous patterns found so far in few clades of terrestrial vertebrates. These results also suggest that vulnerable species accumulate more deleterious substitutions than non-threatened ones, which give insights in how life-history traits, populations sizes and genetic risk of extinction can be tightly interconnected.

molecular biology↗

A strategy for complete telomere-to-telomere assembly of ciliate macronuclear genome using ultra-high coverage Nanopore data

Ciliates contain two kinds of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC) in a single cell. The MAC usually have fragmented chromosomes. These fragmented chromosomes, capped with telomeres at both ends, could be gene size to several megabases in length among different ciliate species. So far, no telomere-to-telomere assembly of entire MAC genome in ciliate species is finished. Development of the third generation sequencing technologies allows to generate sequencing reads up to megabases in length that could possibly span an entire MAC chromosome. Taking advantage of ultra-long Nanopore reads, we established a simple strategy for the complete assembly of ciliate MAC genomes. Using this strategy, we assembled the complete MAC genomes of two ciliate species Tetrahymena thermophila and Tetrahymena shanghaiensis, composed of 181 and 214 chromosomes telomere-to-telomere respectively. The established strategy as well as the high-quality genome data will provide a useful approach for ciliate genome assembly, and a valuable community resource for further biological, evolutionary and population genomic studies.

genomics↗

New Evolutionary and Structural Insights In Plasmodium Proteins

One of the most debated topics in Evolutionary Biology concerns Low Complexity Regions of P. falciparum, the causative agent of the most virulent and deadly form of human malaria. In this work, we analysed the proteome of 22 plasmodium species including P. falciparum. SEG predicts that proteins containing Low Complexity Regions turn out to be longer than those which are predicted to be completely complex (without Low Complexity Regions). Moreover, using some well-known bioinformatics tools such as the Effective Number of Codons, the Pr2 and a new index that we have called SPI, we have noticed how proteins that embed Low Complexity Regions are under lower selective pressure than those that do not present this type of locus. By applying the Relative Synonymous Codon Usage and other tools developed ad hoc for this study, we note, instead, how the Low Complexity Regions appear to have a non-neutral codon bias with respect to the host proteins.

bioinformatics↗

Inspecting abundantly expressed genes in male strobili in sugi (Cryptomeria japonica D. Don) via a highly accurate cDNA assembly

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. The field of functional genomics is rapidly developing. The genomics of this gymnosperm species is currently being studied. Although its genomic size is 11 Gbps, it is still too large to assemble well within a short period of time. Transcriptomics is the one another approach to address this. Moreover, it is a necessary step in obtaining the complete genomic data. Here we designed a three stages assembling workflow using the de novo transcriptome assembly tools, Oases and Trinity. The three stages in transcriptomics are independent assembly, automatic and semi-automatic integration, and refinement by filtering out potential contamination. We found a set of 49,795 cDNA and an equal number of translated proteins (CJ3006NRE). According to the benchmark of BUSCO, 87.01 % were complete genes, including very high "Complete and single-copy" genes-78.47%. Compared to other full-length cDNA resources, the extent of the coverage in CJ3006NRE suggests that it may be used as the standard for further studies. When two tissue-specific libraries were compared, principal component analysis (PCA) showed that there were significant differences between male strobili and leaf and bark sets. The highest three upregulated transcription factors stood out as orthologs to angiosperms. The identified signature-like domain of the transcription factors demonstrated the accuracy of the assembly. Based on the evaluation of different resources, we demonstrate that our transcriptome assembly output is valuable and useful for further studies in functional genomics and evolutionary biology.

bioinformatics↗

Transcriptome analysis reveals the genetic basis underlying the development of skin appendages and immunity in hedgehog (Atelerix albiventris)

The expression of hair features is an evolutionary adaptation resulting from interactions between many organisms and their environment. Elucidation of the mechanisms that underlie the expression of such traits is a topic in evolutionary biology research. Therefore, we assessed the de novo transcriptome of Atelerix albiventris at three developmental stages and compared gene expression profiles between abdomen hair and dorsal spine tissues. We identified 328,576 unigenes in our transcriptome, among which 3,598 were differentially expressed between hair- and spine-type tissues. Dorsal and abdomen skin tissues 5 days after birth were compared and the resulting differentially expressed genes were mainly enriched in keratin filament, epithelium cell differentiation, and epidermis development based on GO enrichment analysis, and tight junction, p53, and cell cycle signaling pathways based on KEGG enrichment analysis. Expression variations of MBP8, SFN, Wnt10, KRT1, and KRT2 may be the main factors regulating hair and spine differentiation for the hedgehog. Strikingly, DEGs in hair-type tissues were also significantly enriched in immune-related terms and pathways with hair-type tissues exhibiting more upregulated immune genes than spine-type tissues. Thus, we propose that spine development was an adaptation that provided protection against injuries or stress and reduced hedgehog vulnerability to infection.

molecular biology↗

Resource availability and availability and disturbance shape maximum tree height across the Amazon

The factors shaping the distribution of giant tropical trees are poorly understood, despite its importance as a link between evolutionary biology and ecosystem biogeochemistry. The recent discovery of clusters of trees over 80 metres tall in the Guiana Shield region of the Amazon rainforest challenges the current understanding of the factors controlling the growth and survival of giant trees. The new discovery led us to revisit the question: what determines the distribution of the tallest trees of the Amazon? Here, we used high-resolution airborne LiDAR (Light Detection and Ranging) surveys to measure canopy height across 282,750 ha of primary old-growth and secondary forests throughout the entire Brazilian Amazon to investigate the relationship between the occurrence of giant trees and the environmental factors that influence their growth and survival. Our results suggest that the factors controlling where trees grow extremely tall are distinct from those controlling their longevity. Trees grow taller in areas with high soil clay content (> 42%), lower radiation (< 130 clear days per year) and wind speeds, avoiding alluvial areas (elevations higher than 40 m a.s.l), and with an optimal precipitation range of 1,500 to 2,500 mm yr-1. We then used an envelope model to determine the environmental conditions that support the very tallest trees (i.e. over 70 m height). We found that, as opposed to the myriad of interacting factors that control the maximum height at a large scale, wind speed had by far the largest influence on the distribution of these sentinel trees, and explained 67% of the probability of finding trees over 70 m in the Brazilian Amazon forest. The high-resolution pan-Amazon LiDAR data showed that environmental variables that drive growth in height are fundamentally different from environmental variables that support their survival. While precipitation and temperature seem to have lower importance for their survival than expected from previous studies, changes in wind and radiation regimes could reshape our forested biomes. This should be carefully considered by policy-makers when identifying important hotspots for the conservation of biodiversity in the Amazon.

ecology↗

Patch-clamp recordings in slices of telencephalon, diencephalon and rhombencephalon of salamanders

AO_SCPLOWBSTRACTC_SCPLOWThe salamander is a key limbed vertebrate from which many major scientific questions can be addressed in the fields of motor control, evolutionary biology, and regeneration biology. An important gap of knowledge is the description of the electrophysiological properties of the neurons constituting their central nervous system. To our knowledge, some patch-clamp electrophysiological recordings were done in the spinal cord and recently in hindbrain slices, but not in any higher brain region. Here, we present a method to obtain patch-clamp recordings in slices of the telencephalon, diencephalon and rhombencephalon of salamanders. The method includes dissection of the brain, brain slice preparation, visual identification of neurons and patch-clamp recordings. We provide single cell recordings in the rhombencephalon, diencephalon and telencephalon of salamanders. This method should open new avenues to dissect the operation of salamander brain circuits at the cellular level. HO_SCPLOWIGHLIGHTSC_SCPLOW- Salamander brain slices of telencephalon, diencephalon, and rhombencephalon - Patch-clamp recordings in salamander brain slices - The salamander as a model to decipher tetrapod neural microcircuits

neuroscience↗

Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling

Human plasma contains >40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from >19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.

genomics↗

Denisovan introgression has shaped the immune system of present-day Papuans

Modern humans have substantially admixed with multiple archaic hominins. Papuans, in particular, owe up to 5% of their genome to Denisovans, a sister group to Neanderthals, whose remains have only been identified in Siberia and Tibet. Unfortunately, the biological and evolutionary significance of these introgression events remain poorly understood. Here we investigate the function of archaic alleles of both Denisovan and Neanderthal ancestry characterised within a previously published set of 56 genomes from individuals of Papuan genetic ancestry living in the island of New Guinea. By comparing the distribution of archaic and modern human variants, we are able to assess the consequences of archaic admixture across a multitude of different cell types and functional elements. We detect a consistent signal across Denisovan variants of strong involvement in immune-related processes throughout our analyses. Archaic alleles are often located within cis-regulatory elements and transcribed regions of the genome, suggesting that they are capable of contributing to a wide range of cellular regulatory processes. We identify 3,538 high-confidence Denisovan variants that fall within annotated cis-regulatory elements and have the potential to alter the affinity of multiple transcription factors to their cognate DNA motifs, highlighting a likely mechanism by which introgressed DNA can impact phenotypes in present-day humans. Lastly, we experimentally validate these predictions by testing the regulatory potential of five Denisovan variants segregating at high frequency within Papuan individuals, and find that two are associated with a significant reduction of transcriptional activities in plasmid reporter assays relative to modern human alleles. Together, these data provide support for the hypothesis that, despite their broadly deleterious nature, archaic alleles actively contribute to modern human phenotypic diversity to this day, and might have facilitated early adaptation to non-African environments.

evolutionary biology↗

Exact decoding of the sequentially Markov coalescent

In statistical genetics, the sequentially Markov coalescent (SMC) is an important framework for approximating the distribution of genetic variation data under complex evolutionary models. Methods based on SMC are widely used in genetics and evolutionary biology, with significant applications to genotype phasing and imputation, recombination rate estimation, and inferring population history. SMC allows for likelihood-based inference using hidden Markov models (HMMs), where the latent variable represents a genealogy. Because genealogies are continuous, while HMMs are discrete, SMC requires discretizing the space of trees in a way that is complicated and can lead to bias. In this work, we propose a method that circumvents this requirement, enabling SMC-based inference to be performed in the natural setting of a continuous state space. We derive fast, exact methods for frequentist and Bayesian inference using SMC. Compared to existing methods, ours requires minimal user intervention or parameter tuning, no numerical optimization or E-M, and is faster and more accurate.

genetics↗

Evidence for Numerous Embedded Antisense Overlapping Genes in Diverse E. coli Strains

The genetic code allows six reading frames at a double-stranded DNA locus, and many open reading frames (ORFs) overlap extensively with ORFs of annotated genes (e.g., at least 30 bp or having an embedded ORF). Currently, bacterial genome annotation systematically discards embedded overlapping ORFs of genes (OLGs) due to an assumed information-content constraint, and, consequently, very few OLGs are known. Here we use strand-specific RNAseq and ribosome profiling, detecting about 200 embedded or partially overlapping ORFs of gene candidates in the pathogen E. coli O157:H7 EDL933. These are typically short, many of them show clear promoter motifs as determined by Cappable-seq, indistinguishable from those of annotated genes, and are expressed at a low level. We could express most of them as stable proteins, and 49 displayed a potential phenotype. Ribosome profiling analyses in three other E. coli strains predicted between 84 and 190 embedded antisense OLGs per strain except in E. coli K-12, which is an atypical lab strain. We also found evidence of homology to annotated genes for 100 to 300 OLGs per E. coli strain investigated. Based on this evidence we suggest that bacterial OLGs deserve attention with respect to genome annotation and coding complexity of bacterial genomes. Such sequences may constitute an important coding reserve, opening up new research in genetics and evolutionary biology.

microbiology↗

A highly contiguous genome assembly of a major forest pest, the Eurasian spruce bark beetle Ips typographus

The Eurasian spruce bark beetle (Ips typographus [L.]), is a major killer of spruce forests across the Palearctic. During epidemics, it can destroy over 100 million cubic meters of spruce trees in a single year. Here we report a 236 Mb, highly contiguous I. typographus genome assembly using PacBio long-read sequencing. The final phased assembly had a contig N50 of 6.65 Mb in 272 contigs and was predicted to contain 23,923 protein-coding genes. Comparative genomic analysis revealed expanded gene families associated with plant cell wall degradation, including pectinases, aspartyl proteases, and glycosyl hydrolases. In todays forests, increasingly stressed by global warming, this resource can assist in mitigating bark beetle outbreaks by developing novel pest control strategies. Further, this first whole-genome sequence from the genus Ips provides timely resources to address important questions about the evolutionary biology and ecology of Curculionidae, the true weevils, one of the largest animal families.

genomics↗

Conserved and unique transcriptional features of pharyngeal arches in the skate (Leucoraja erinacea) and evolution of the jaw

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers bapx1 and gdf5 and pro-chondrogenic transcription factors barx1 and gsc, and the dorsalising transcription factor pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

developmental biology↗

Genetic diversity, population structure and selection signature in Ethiopian Sorghum (Sorghum bicolor L. ) germplasm

Ethiopia, the probable center of origin and diversity for sorghum (Sorghum bicolor L. [Moench]) and with unique eco-geographic features, possesses a large number of sorghum landraces that have not been well studied. Increased knowledge of this diverse germplasm through large-scale genomic characterization may contribute for understanding of evolutionary biology, and adequate use of these valuable resources from the center of origin. In this study, we characterized genetic diversity, population structure and selection signature in 304 sorghum accessions collected from diverse sorghum growing regions of Ethiopia using genotyping-by-sequencing (GBS). We identified a total of 108,107 high-quality single nucleotide polymorphism (SNPs) markers that were evenly distributed across the sorghum genome. The average gene diversity among accessions was high (He = 0.29). We detected a relatively low frequency of rare alleles (26%), highlighting the potential of this germplasm for subsequent allele mining studies through genome wide association studies (GWAS). While we found no evidence of genetic differentiation among administrative regions (FST = 0.02, p = 0.12), population structure and cluster analyses showed clear differentiation among six Ethiopian sorghum populations (FST = 0.28, p = 0.01) adapting to different environments. Analysis of SNP differentiation between the identified genetic groups revealed a total of 40 genomic regions carrying signatures of selection. These regions harbored candidate genes potentially involved in a variety of biological processes, including abiotic stress tolerance, pathogen defense and reproduction. Overall, a high level of untapped diversity for sorghum improvement remains available in Ethiopia, with patterns of diversity consistent with divergent selection on a range of adaptive characteristics.

genetics↗

Variant Library Annotation Tool (VaLiAnT): an oligonucleotide library design and annotation tool for Saturation Genome Editing and other Deep Mutational Scanning experiments

MotivationRecent advances in CRISPR/Cas9 technology allow for the functional analysis of genetic variants at single nucleotide resolution whilst maintaining genomic context (Findlay et al., 2018). This approach, known as saturation genome editing (SGE), is a distinct type of deep mutational scanning (DMS) that systematically alters each position in a target region to explore its function. SGE experiments require the design and synthesis of oligonucleotide variant libraries which are introduced into the genome by homology-directed repair (HDR). This technology is broadly applicable to diverse research fields such as disease variant identification, drug development, structure-function studies, synthetic biology, evolutionary genetics and the study of host-pathogen interactions. Here we present the Variant Library Annotation Tool (VaLiAnT) which can be used to generate saturation mutagenesis oligonucleotide libraries from user-defined genomic coordinates and standardised input files. This software package is intentionally versatile to accommodate diverse operability, with species, genomic reference sequences and transcriptomic annotations specified by the user. Genomic ranges, directionality and frame information are considered to allow perturbations at both the nucleotide and amino acid level. ResultsCoordinates for a genomic range, that may include exonic and/or intronic sequence, are provided by the user in order to retrieve a corresponding oligonucleotide reference sequence. A user-specified range within this sequence is then subject to systematic, nucleotide and/or amino acid saturating mutator functions, with each discrete mutation returned to the user as a separate sequence, building up the final oligo library. If desired, variant accessions from genetic information repositories, such as ClinVar and gnomAD, that fall within the user-specified ranges, will also be incorporated into the library. For SGE library generation, base reference sequences can be modified to include PAM (Protospacer Adjacent Motif) and protospacer protection edits that prevent Cas9 from cutting incorporated oligonucleotide tracts. Mutator functions modify this protected reference sequence to generate variant sequences. Constant regions are designated for non-editing to allow specific adapter annealing for downstream cloning and amplification from the library pool. A metadata file is generated, delineating annotation information for each variant sequence to aid computational analysis. In addition, a library file is generated, which contains unique sequences (any exact duplicate sequences are removed) ready for submission to commercial synthesis platforms. A VCF file listing all variants is also generated for analysis and quality control processes. The VaLiAnT software package provides a novel means to systemically retrieve, mutate and annotate genomic sequences for oligonucleotide library generation. Specific features for SGE library generation can be employed, with other diverse applications possible. Availability and ImplementationVaLiAnT is a command line tool written in Python. Source code, testing data, example library input and output files, and executables are available at https://github.com/cancerit/VaLiAnT. A user manual details step by step instructions for software use, available at https://github.com/cancerit/VaLiAnT/wiki. The software is freely available for non-commercial use (see Licence for more details, https://github.com/cancerit/VaLiAnT/blob/develop/LICENSE).

genomics↗

Testing for parallel genomic and epigenomic footprints of adaptation to urban life in a passerine bird

Identifying the molecular mechanisms involved in rapid adaptation to novel environments and determining their predictability are central questions in Evolutionary Biology and pressing issues due to rapid global changes. Complementary to genetic responses to selection, faster epigenetic variations such as modifications of DNA methylation may play a substantial role in rapid adaptation. In the context of rampant urbanization, joint examinations of genomic and epigenomic mechanisms are still lacking. Here, we investigated genomic (SNP) and epigenomic (CpG methylation) responses to urban life in a passerine bird, the Great tit (Parus major). To test whether urban evolution is predictable (i.e parallel) or involves mostly non-parallel molecular processes among cities, we analysed three distinct pairs of city and forest Great tit populations across Europe. Results reveal a polygenic response to urban life, with both many genes putatively under weak divergent selection and multiple differentially methylated regions (DMRs) between forest and city great tits. DMRs mainly overlapped transcription start sites and promotor regions, suggesting their importance in the modulation gene expression. Both genomic and epigenomic outliers were found in genomic regions enriched for genes with biological functions related to nervous system, immunity, behaviour, hormonal and stress responses. Interestingly, comparisons across the three pairs of city-forest populations suggested little parallelism in both genetic and epigenetic responses. Our results confirm, at both the genetic and epigenetic levels, hypotheses of polygenic and largely non-parallel mechanisms of rapid adaptation in new environments such as urbanized areas.

genomics↗