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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Comprehensive profiling of TCRβ repertoire in a non-model species (the bank vole) using high-throughput sequencing

In recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. We present the first in-depth characterization of the TCR{beta} repertoire in a non-model mammal with limited genomic resources available - the bank vole (Myodes glareolus). We used 5'RACE and Illumina HTS to describe V and J segments and to qualitatively characterize preferential V-J segment usage and CDR3 length distribution. Finally, a molecular protocol integrating unique molecular identifiers was used for quantitative analysis of CDR3 repertoire with stringent error correction. We found 37 V and 11 J genes that were orthologous to mice genes. A conservative, lower bound estimation of the TCR{beta} repertoire was 1.7-2.3x105 clonotypes, and the degree of sharing of the observed repertoire between any two individuals was 3.6% of nucleotide sequences and 14.3% of amino acid sequences. Our work adds a crucial element to the immunogenetic resources available for the bank vole, an important species in ecological and evolutionary research. The workflow that we developed can be applied for immune repertoire sequencing of non-model species, including endangered vertebrates.

immunology↗

bioRxiv2017

Don’t throw out the sympatric species with the crater lake water: fine-scale investigation of introgression provides weak support for functional role of secondary gene flow in one of the clearest examples of sympatric speciation

Genomic data has revealed complex histories of colonization and repeated gene flow previously unrecognized in some of the most celebrated examples of sympatric speciation and radiation. However, much of the evidence for secondary gene flow into these radiations comes from genome-wide tests, which tells us little about how gene flow potentially influenced sympatric diversification. Here we investigated whole genomes of Barombi Mbo crater lake cichlids for fine-scale patterns of introgression between species with neighboring riverine cichlid populations. We did find evidence of secondary gene flow into the radiation scattered across < 0.24% of the genome; however, the functional and genetic diversity in these regions paint no clear picture of how that variation could have contributed to the ecological and morphological diversity found in the lake. Our results suggest that either variation in novel genetic pathways introduced during secondary gene flow contributed to the radiation, or that secondary gene flow was predominantly neutral with respect to the diversification processes. We also found evidence for differential assortment of ancestral polymorphism found in riverine populations between sympatric sister species, suggesting the presence of a hybrid swarm in the past. While the history of gene flow and colonization appears to be more complicated than once thought, the lack of compelling evidence for secondary gene flow influencing diversification suggests that we should not yet rule out one of the most celebrated examples of sympatric speciation in nature.

evolutionary biology↗

bioRxiv2017

The unrealized potential of herbaria in global change biology

Plant and fungal specimens in herbaria are becoming primary resources for investigating how plant phenology and geographic distributions shift with climate change, greatly expanding inferences across spatial, temporal, and phylogenetic dimensions. However, these specimens contain a wealth of additional data--including nutrients, defensive compounds, herbivore damage, disease lesions, and signatures of physiological processes--that capture ecological and evolutionary responses to the Anthropocene but which are less frequently utilized. Here, we outline the diversity of herbarium data, global change topics to which they have been applied, and new hypotheses they could inform. We find that herbarium data have been used extensively to study impacts of climate change and invasive species, but that such data are less commonly used to address other drivers of biodiversity loss, including habitat conversion, pollution, and overexploitation. In addition, we note that fungal specimens are under-explored relative to vascular plants. To facilitate broader application of plant and fungal specimens in global change research, we outline the limitations of these data and modern sampling and statistical tools that may be applied to surmount challenges they present. Using a case study of insect herbivory, we illustrate how novel herbarium data may be employed to test hypotheses for which few data exist, despite potentially large biases. With the goal of positioning herbaria as hubs for global change research, we suggest future research directions and curation priorities.

plant biology↗

bioRxiv2017

Balances: a new perspective for microbiome analysis

High-throughput sequencing technologies have revolutionized microbiome research by allowing the relative quantification of microbiome composition and function in different environments. One of the main goals in microbiome analysis is the identification of microbial species that are differentially abundant among groups of samples, or whose abundance is associated with a variable of interest. Most available methods for microbiome abundance testing perform univariate tests for each microbial species or taxa separately, ignoring the compositional nature of microbiome data.\n\nWe propose an alternative approach for microbiome abundance testing that consists on the identification of two groups of taxa whose relative abundance, or balance, is associated with the response variable of interest. This approach is appealing, since it has direct translation to the biological concept of ecological balance between species in an ecosystem. In this work, we present selbal, a greedy stepwise algorithm for balance selection. We illustrate the algorithm with 16s abundance data from an HIV-microbiome study and a Crohn-microbiome study.\n\nImportanceA more meaningful approach for microbiome abundance testing is presented. Instead of testing each taxon separately we propose to explore abundance balances among groups of taxa. This approach acknowledges the compositional nature of microbiome data.

bioinformatics↗

bioRxiv2017

Novel bacterial lineages associated with boreal moss species

Mosses are critical components of boreal ecosystems where they typically account for a large proportion of net primary productivity and harbor diverse bacterial communities that can be the major source of biologically-fixed nitrogen in these ecosystems. Despite their ecological importance, we have limited understanding of how microbial communities vary across boreal moss species and the extent to which local environmental conditions may influence the composition of these bacterial communities. We used marker gene sequencing to analyze bacterial communities associated with eight boreal moss species collected near Fairbanks, AK USA. We found that host identity was more important than site in determining bacterial community composition and that mosses harbor diverse lineages of potential N2- fixers as well as an abundance of novel taxa assigned to understudied bacterial phyla (including candidate phylum WPS-2). We performed shotgun metagenomic sequencing to assemble genomes from the WPS-2 candidate phylum and found that these moss-associated bacteria are likely anoxygenic phototrophs capable of carbon fixation via RuBisCo with an ability to utilize byproducts of photorespiration from hosts via a glyoxylate shunt. These results give new insights into the metabolic capabilities of understudied bacterial lineages that associate with mosses and the importance of plant hosts in shaping their microbiomes.

microbiology↗

bioRxiv2017

Metaproteomics of colonic microbiota unveils discrete protein functions among colitic mice and control groups

Metaproteomics can greatly assist established high-throughput sequencing methodologies to provide systems biological insights into the alterations of microbial protein functionalities correlated with disease-associated dysbiosis of the intestinal microbiota. Here, we utilized the well-characterized murine T cell transfer model of colitis to find specific changes within the intestinal luminal proteome associated with inflammation. MS proteomic analysis of colonic samples permitted the identification of {bsim}10,000-12,000 unique peptides that corresponded to 5,610 protein clusters identified across three groups, including the colitic Rag1-/- T cell recipients, isogenic Rag1-/- controls, and wild-type mice. We demonstrate that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia and show that such alterations in the microbial communities contributed to the enrichment of specific proteins with transcription and translation gene ontology terms. In combination with 16S sequencing, our metaproteomics-based microbiome studies provide a foundation for assessing alterations in intestinal luminal protein functionalities in a robust and well-characterized mouse model of colitis, and set the stage for future studies to further explore the functional mechanisms of altered protein functionalities associated with dysbiosis and inflammation.\n\nStatement of significance of the studyThe commensal gut microbiota is essential to maintaining health and has a primary role in digestion/metabolism, homeostasis, and protection from pathogenic bacteria. High-throughput sequencing has established Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria as the four major bacterial phyla that comprise the ecological makeup of the intestinal microbiota. However, the tremendous inter-/intra-variability in microbial composition across individuals, as well as along the length of the intestinal tract has made it difficult to definitively ascertain specific bacterial species associated with health or as drivers of disease states, including inflammatory bowel disease. In this study, we expanded upon the current metaproteomics techniques and use the robust and highly reproducible murine T cell transfer model of colitis as well as a comprehensive database of mouse, human, plant, and all microbial genomes sequenced to date to elucidate alterations in both host and gut microbial proteins associated with intestinal inflammation. Our results show that host genetics, gut microbiota, and inflammation have tremendous influences on the intestinal luminal proteomic landscape.

bioinformatics↗

bioRxiv2017

Fungal communities associated with the eastern larch beetle: diversity and variation within developmental stages

Bacterial and fungal communities associated with bark beetles, especially some Dendroctonus species, mediate challenging aspects of the subcortical habitat for their hosts. Filamentous fungi and yeasts are important in various metabolic processes of certain bark beetles, with involvement in nutrition, protection and detoxification of plant defense compounds. The eastern larch beetle, Dendroctonus simplex, is recognized as a serious forest pest in the upper part of North America. Although D. simplex is well studied, the fungal communities and their ecological role remain to be investigated. Here, using 18S rRNA gene pyrosequencing, we provide a comprehensive overview of the yeasts and filamentous fungi associated with the eastern larch beetle and compare fungal communities between different developmental stages and microenvironments. Fungal mycobiome associated with the galleries was also investigated. Our study has unveiled an unexpected fungal diversity associated with the developmental stages. Significant differences in species richness between the developmental stages were determined. Yeasts were found to be predominant in the adult and larval stages, whereas filamentous fungi were most prevalent in the pupae. Our results indicate a possible implication of yeasts in the eastern larch beetle nutrition.

microbiology↗

bioRxiv2017

The study of animal behaviour as a physical science

Behaviour is the ultimate output of an animals nervous system and choosing the right action at the right time can be critical for survival. The study of the organisation of behaviour in its natural context, ethology, has historically been a primarily qualitative science. A quantitative theory of behaviour would advance research in neuroscience as well as ecology and evolution. However, animal posture typically has many degrees of freedom and behavioural dynamics vary on timescales ranging from milliseconds to years, presenting both technical and conceptual challenges. Here we review 1) advances in imaging and computer vision that are making it possible to capture increasingly complete records of animal motion and 2) new approaches to understanding the resulting behavioural data sets. With the right analytical approaches, these data are allowing researchers to revisit longstanding questions about the structure and organisation of animal behaviour and to put unifying principles on a quantitative footing. Contributions from both experimentalists and theorists are leading to the emergence of a physics of behaviour and the prospect of discovering laws and developing theories with broad applicability. We believe that there now exists an opportunity to develop theories of behaviour which can be tested using these data sets leading to a deeper understanding of how and why animals behave.

animal behavior and cognition↗

bioRxiv2017

Improved genome assembly and annotation for the rock pigeon (Columba livia)

The domestic rock pigeon (Columba livia) is among the most widely distributed and phenotypically diverse avian species. This species is broadly studied in ecology, genetics, physiology, behavior, and evolutionary biology, and has recently emerged as a model for understanding the molecular basis of anatomical diversity, the magnetic sense, and other key aspects of avian biology. Here we report an update to the C. livia genome reference assembly and gene annotation dataset. Greatly increased scaffold lengths in the updated reference assembly, along with an updated annotation set, provide improved tools for evolutionary and functional genetic studies of the pigeon, and for comparative avian genomics in general.

genomics↗

bioRxiv2017

Infino: a Bayesian hierarchical model improves estimates of immune infiltration into tumor microenvironment

Robust quantification of immune cell infiltration into the tumor microenvironment may shed light on why only a small proportion of patients benefit from checkpoint therapy. The immune cells surrounding a tumor have been suggested to mediate an effective response to immunotherapy. However, traditional measurement of immune cell content around a tumor by immunohistochemistry, flow cytometry, or mass cytometry allows measurement of only up to a few dozen markers at a time, limiting the number of immune cell types identified. Immune cell type abundances may instead be estimated in silico by deconvolving gene expression mixtures from bulk RNA sequencing of tumor tissue. By measuring tens of thousands of transcripts at once, bulk RNA-seq provides a rich input to algorithms that quantify cell type abundances in the tumor microenvironment, affording the potential to quantify the states of a greater number of immune cell types (given adequate training data). Here, we first review existing methods for deconvolution and evaluate their performance on synthetic mixtures. Then we develop a Bayesian inference approach, named infino, that learns to distinguish immune cell expression phenotypes and deconvolve mixtures. In contrast to earlier approaches, infino accepts RNA sequencing data, models transcript expression variability, and exploits the relationships between cell types to improve deconvolution accuracy and allow interrogation from the level of broad categories to the level of finest granularity. The resulting probability distributions of immune infiltration could be applied to numerous questions concerning the diverse ecology of immune cell types, including assessment of the association of immune infiltration with response to immunotherapy, and study of the expression profile and presence of elusive T cell subcompartments, such as T cell exhaustion.

bioinformatics↗

bioRxiv2017

Endospores and other lysis-resistant bacteria comprise a widely shared core community within the human microbiota

Endospore-formers in the human microbiota are well adapted for host-to-host transmission, and an emerging consensus points to their role in determining health and disease states in the gut. The human gut, more than any other environment, encourages the maintenance of endospore formation, with recent culture-based work suggesting that over 50% of genera in the microbiome carry genes attributed to this trait. However, there has been limited work on the ecological role of endospores and other stress-resistant cellular states in the human gut. In fact, there is no data to indicate whether organisms with the genetic potential to form endospores actually form endospores in situ and how sporulation varies across individuals and over time. Here, we applied a culture-independent protocol to enrich for endospores and other stress-resistant cells in human feces to identify variation in these states across people and within an individual over time. We see that cells with resistant states are more likely than those without to be shared among multiple individuals, which suggests that these resistant states are particularly adapted for cross-host dissemination. Furthermore, we use untargeted fecal metabolomics in 24 individuals and within a person over time to show that these organisms respond to shared environmental signals, and in particular, dietary fatty acids, that likely mediate colonization of recently disturbed human guts.

microbiology↗

bioRxiv2017

Bacterial adaptation to host diet is a key evolutionary force shaping host-microbe symbiosis

Life on Earth was dominated by bacteria for billions of years1. About 600 million years ago, animal life emerged and micro-organisms played a crucial role in shaping animal development, physiology and evolution2-4. The two partners committed to a symbiotic relationship that persists in nearly all animals today. Such beneficial interactions are pervasive throughout nature and have been extensively characterized5,6. However, the ecological and evolutionary forces that drive the emergence and evolution of the symbiont benefits to their animal hosts remain largely elusive. Here we show that the host nutritional environment, instead of the host, is a predominant driving force in this evolutionary process and we identify a mechanism resulting from the bacterial adaptation to the diet, which confers improved functional benefit to the host. By applying experimental evolution to a model of host-bacteria beneficial symbiosis: Drosophila melanogaster associated with Lactobacillus plantarum, one of its growth promoting symbiotic bacteria7,8, we found that the de novo mutations in the same acetate kinase (ackA) locus invariably emerge first, rapidly become fixed, and such evolution occurs with or without the host. Furthermore, we demonstrate that ackA mutations trigger the increased production of N-acetyl-glutamine, which is sufficient to confer improved host growth capabilities to the evolved bacterial strains. Our study therefore identifies a specific mechanism by which a symbiotic bacterium increases its benefit to its animal host and reveals that adaptation to the host diet is a foremost step in the determination of the evolutionary course of symbiosis between an animal and its gut microbes.

evolutionary biology↗

bioRxiv2017

Computational haplotype recovery and long-read validation identifies novel isoforms of industrially relevant enzymes from natural microbial communities

Elucidation of population-level diversity of microbiomes is a significant step towards a complete understanding of the evolutionary, ecological and functional importance of microbial communities. Characterizing this diversity requires the recovery of the exact DNA sequence (haplotype) of each gene isoform from every individual present in the community. To address this, we present Hansel and Gretel: a freely-available data structure and algorithm, providing a software package that reconstructs the most likely haplotypes from metagenomes. We demonstrate recovery of haplotypes from short-read Illumina data for a bovine rumen microbiome, and verify our predictions are 100% accurate with long-read PacBio CCS sequencing. We show that Gretels haplotypes can be analyzed to determine a significant difference in mutation rates between core and accessory gene families in an ovine rumen microbiome. All tools, documentation and data for evaluation are open source and available via our repository: https://github.com/samstudio8/gretel

bioinformatics↗

bioRxiv2017

Genome-wide detection of genes under positive selection in worldwide populations of the barley scald pathogen

The coevolution between hosts and pathogens generates strong selection pressures to maintain resistance and infectivity, respectively. Genomes of plant pathogens often encode major effect loci for the ability to successfully infect a specific host. Hence, heterogeneity in the host genotypes and abiotic factors leads to locally adapted pathogen populations. However, the genetic basis of local adaptation is poorly understood. We analyzed global field populations of Rhynchosporium commune, the pathogen causing barley scald disease, to identify candidate genes for local adaptation. Whole genome sequencing data generated for 125 isolates showed that the pathogen is subdivided into three genetic clusters associated with distinct geographic and climatic regions. Using haplotype-based selection scans applied independently to each genetic cluster, we found strong evidence for selective sweeps throughout the genome. Comparisons of loci under selection among clusters revealed little overlap, suggesting that ecological differences associated with each cluster led to variable selection regimes. The strongest signals of selection were found predominantly in the two clusters composed of isolates from Central Europe and Ethiopia. The strongest selective sweep regions encoded proteins with functions related to both biotic and abiotic stresses. We found that selective sweep regions were enriched in genes encoding functions in cellular localization, protein transport activity, and DNA damage responses. In contrast to the prevailing view that a small number of gene-for-gene interactions govern plant pathogen evolution, our analyses suggest that the evolutionary trajectory is largely determined by spatially heterogeneous biotic and abiotic selection pressures.

genomics↗

bioRxiv2017

Pathogen evolution: slow and steady spreads the best

The theory of life history evolution provides a powerful framework to understand the evolutionary dynamics of pathogens in both epidemic and endemic situations. This framework, however, relies on the assumption that pathogen populations are very large and that one can neglect the effects of demographic stochasticity. Here we expand the theory of life history evolution to account for the effects of finite population size on the evolution of pathogen virulence. We show that demographic stochasticity introduces additional evolutionary forces that can qualitatively affect the dynamics and the evolutionary outcome. We discuss the importance of the shape of pathogen fitness landscape and host heterogeneity on the balance between mutation, selection and genetic drift. In particular, we discuss scenarios where finite population size can dramatically affect classical predictions of deterministic models. This analysis reconciles Adaptive Dynamics with population genetics in finite populations and thus provides a new theoretical toolbox to study life-history evolution in realistic ecological scenarios.

evolutionary biology↗

bioRxiv2017

A quantitative PCR based environmental DNA assay for detecting Atlantic salmon (Salmo salar L.)

O_LIThe Atlantic salmon (Salmo salar L.) has worldwide ecological, cultural and economic importance. The species has undergone extensive decline across its native range, yet concerns have been raised about its invasive potential in the Pacific. Knowledge on the distribution of this species is vital for addressing conservation goals.\nC_LIO_LIThis study presents an eDNA assay to detect S. salar in water samples, using quantitative PCR (qPCR) technology. Species-specific primers and a minor groove binding (MGB) probe were designed for the assay, based on the mitochondrial cytochrome oxidase I (COI) gene.\nC_LIO_LIThe results of this study indicate that eDNA is a highly sensitive tool for detecting S. salar in situ, and could potentially provide an alternative, non-invasive method for determining the distribution of this species.\nC_LI

genetics↗

bioRxiv2017

A 16S rRNA gene sequencing and analysis protocol for the Illumina MiniSeq platform

High-throughput sequencing of the 16S rRNA gene is widely used in microbial ecology, with Illumina platforms being widely used in recent studies. The MiniSeq, Illuminas latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger sequencing platforms (e.g. MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed an additional sequencing primer that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples as a quality control benchmark. After careful filtering procedures, we were able to recapture a realistic richness of the mock community, and identify meaningful differences in alpha and beta diversity in the environmental samples. These results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.\n\nIMPORTANCEWe modified a custom sequencing approach and used a mock community to test the fidelity of high-throughput sequencing on the Illumina MiniSeq platform. Our results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq. In addition, our protocol increases feasibility for small laboratories to perform their own high-throughput sequencing of the 16S rRNA marker gene.

microbiology↗

bioRxiv2017

ANTISEPTIC AGENTS ELICIT SHORT-TERM, PERSONALIZED AND BODY SITE-SPECIFIC SHIFTS IN RESIDENT SKIN BACTERIAL COMMUNITIES

Despite critical functions in cutaneous health and disease, it is unclear how resident skin microbial communities are altered by topical antimicrobial interventions commonly used in personal and clinical settings. Here we show that acute exposure to antiseptic treatments elicits rapid but short-term depletion of microbial community diversity and membership. Thirteen subjects were enrolled in a longitudinal treatment study to analyze the effects of topical treatments (ethanol, povidone-iodine, chlorhexidine, water) on the skin microbiome at two skin sites of disparate microenvironment: forearm and back. Treatment effects were highly dependent on personalized and body site-specific colonization signatures, which concealed community dynamics at the population level when not accounted for in this analysis. The magnitude of disruption was influenced by the identity and abundance of particular bacterial inhabitants. Lowly abundant members of the skin microbiota were more likely to be displaced, and subsequently replaced by the most abundant taxa prior to treatment. Members of the skin commensal family Propionibactericeae were particularly resilient to treatment, suggesting a distinct competitive advantage in the face of disturbance. These results provide insight into the stability and resilience of the skin microbiome, while establishing the impact of topical antiseptic treatment on skin bacterial dynamics and community ecology.

microbiology↗