Advancing the analysis of bisulfite sequencing data in its application to ecological plant epigenetics

The aim of this thesis is to bridge the gap between the state-of-the-art bioinformatic tools and resources, currently at the forefront of epigenetic analysis, and their emerging applications to non-model species in the context of plant ecology. New, high-resolution research tools are presented; first in a specific sense, by providing new genomic resources for a selected non-model plant species, and also in a broader sense, by developing new software pipelines to streamline the analysis of bisulfite sequencing data, in a manner which is applicable to a wide range of non-model plant species. The selected species is the annual field pennycress, Thlaspi arvense, which belongs in the same lineage of the Brassicaceae as the closely-related model species, Arabidopsis thaliana, and yet does not benefit from such extensive genomic resources. It is one of three key species in a Europe-wide initiative to understand how epigenetic mechanisms contribute to natural variation, stress responses and long-term adaptation of plants. To this end, this thesis provides a high-quality, chromosome-level assembly for T. arvense, alongside a rich complement of feature annotations of particular relevance to the study of epigenetics. The genome assembly encompasses a hybrid approach, involving both PacBio continuous long reads and circular consensus sequences, alongside Hi-C sequencing, PCR-free Illumina sequencing and genetic maps. The result is a significant improvement in contiguity over the existing draft state from earlier studies. Much of the basis for building an understanding of epigenetic mechanisms in non-model species centres around the study of DNA methylation, and in particular the analysis of bisulfite sequencing data to bring methylation patterns into nucleotide-level resolution. In order to maintain a broad level of comparison between T. arvense and the other selected species under the same initiative, a suite of software pipelines which include mapping, the quantification of methylation values, differential methylation between groups, and epigenome-wide association studies, have also been developed. Furthermore, presented herein is a novel algorithm which can facilitate accurate variant calling from bisulfite sequencing data using conventional approaches, such as FreeBayes or Genome Analysis ToolKit (GATK), which until now was feasible only with specifically-adapted software. This enables researchers to obtain high-quality genetic variants, often essential for contextualising the results of epigenetic experiments, without the need for additional sequencing libraries alongside. Each of these aspects are thoroughly benchmarked, integrated to a robust workflow management system, and adhere to the principles of FAIR (Findability, Accessibility, Interoperability and Reusability). Finally, further consideration is given to the unique difficulties presented by population-scale data, and a number of concepts and ideas are explored in order to improve the feasibility of such analyses. In summary, this thesis introduces new high-resolution tools to facilitate the analysis of epigenetic mechanisms, specifically relating to DNA methylation, in non-model plant data. In addition, thorough benchmarking standards are applied, showcasing the range of technical considerations which are of principal importance when developing new pipelines and tools for the analysis of bisulfite sequencing data. The complete “Epidiverse Toolkit” is available at https://github.com/EpiDiverse and will continue to be updated and improved in the future.:ABSTRACT ACKNOWLEDGEMENTS 1 INTRODUCTION 1.1 ABOUT THIS WORK 1.2 BIOLOGICAL BACKGROUND 1.2.1 Epigenetics in plant ecology 1.2.2 DNA methylation 1.2.3 Maintenance of 5mC patterns in plants 1.2.4 Distribution of 5mC patterns in plants 1.3 TECHNICAL BACKGROUND 1.3.1 DNA sequencing 1.3.2 The case for a high-quality genome assembly 1.3.3 Sequence alignment for NGS 1.3.4 Variant calling approaches 2 BUILDING A SUITABLE REFERENCE GENOME 2.1 INTRODUCTION 2.2 MATERIALS AND METHODS 2.2.1 Seeds for the reference genome development 2.2.2 Sample collection, library preparation, and DNA sequencing 2.2.3 Contig assembly and initial scaffolding 2.2.4 Re-scaffolding 2.2.5 Comparative genomics 2.3 RESULTS 2.3.1 An improved reference genome sequence 2.3.2 Comparative genomics 2.4 DISCUSSION 3 FEATURE ANNOTATION FOR EPIGENOMICS 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS 3.2.1 Tissue preparation for RNA sequencing 3.2.2 RNA extraction and sequencing 3.2.3 Transcriptome assembly 3.2.4 Genome annotation 3.2.5 Transposable element annotations 3.2.6 Small RNA annotations 3.2.7 Expression atlas 3.2.8 DNA methylation 3.3 RESULTS 3.3.1 Transcriptome assembly 3.3.2 Protein-coding genes 3.3.3 Non-coding loci 3.3.4 Transposable elements 3.3.5 Small RNA 3.3.6 Pseudogenes 3.3.7 Gene expression atlas 3.3.8 DNA Methylation 3.4 DISCUSSION 4 BISULFITE SEQUENCING METHODS 4.1 INTRODUCTION 4.2 PRINCIPLES OF BISULFITE SEQUENCING 4.3 EXPERIMENTAL DESIGN 4.4 LIBRARY PREPARATION 4.4.1 Whole Genome Bisulfite Sequencing (WGBS) 4.4.2 Reduced Representation Bisulfite Sequencing (RRBS) 4.4.3 Target capture bisulfite sequencing 4.5 BIOINFORMATIC ANALYSIS OF BISULFITE DATA 4.5.1 Quality Control 4.5.2 Read Alignment 4.5.3 Methylation Calling 4.6 ALTERNATIVE METHODS 5 FROM READ ALIGNMENT TO DNA METHYLATION ANALYSIS 5.1 INTRODUCTION 5.2 MATERIALS AND METHODS 5.2.1 Reference species 5.2.2 Natural accessions 5.2.3 Read simulation 5.2.4 Read alignment 5.2.5 Mapping rates 5.2.6 Precision-recall 5.2.7 Coverage deviation 5.2.8 DNA methylation analysis 5.3 RESULTS 5.4 DISCUSSION 5.5 A PIPELINE FOR WGBS ANALYSIS 6 THERE AND BACK AGAIN: INFERRING GENOMIC INFORMATION 6.1 INTRODUCTION 6.1.1 Implementing a new approach 6.2 MATERIALS AND METHODS 6.2.1 Validation datasets 6.2.2 Read processing and alignment 6.2.3 Variant calling 6.2.4 Benchmarking 6.3 RESULTS 6.4 DISCUSSION 6.5 A PIPELINE FOR SNP VARIANT ANALYSIS 7 POPULATION-LEVEL EPIGENOMICS 7.1 INTRODUCTION 7.2 CHALLENGES IN POPULATION-LEVEL EPIGENOMICS 7.3 DIFFERENTIAL METHYLATION 7.3.1 A pipeline for case/control DMRs 7.3.2 A pipeline for population-level DMRs 7.4 EPIGENOME-WIDE ASSOCIATION STUDIES (EWAS) 7.4.1 A pipeline for EWAS analysis 7.5 GENOTYPING-BY-SEQUENCING (EPIGBS) 7.5.1 Extending the epiGBS pipeline 7.6 POPULATION-LEVEL HAPLOTYPES 7.6.1 Extending the EpiDiverse/SNP pipeline 8 CONCLUSION APPENDICES A. SUPPLEMENT: BUILDING A SUITABLE REFERENCE GENOME B. SUPPLEMENT: FEATURE ANNOTATION FOR EPIGENOMICS C. SUPPLEMENT: FROM READ ALIGNMENT TO DNA METHYLATION ANALYSIS D. SUPPLEMENT: INFERRING GENOMIC INFORMATION BIBLIOGRAPH

Not Always Asthma: Clinical and Legal Consequences of Delayed Diagnosis of Laryngotracheal Stenosis

Laryngotracheal stenosis (LTS) is a rare condition that occurs most commonly as a result of instrumentation of the airway but may also occur as a result of inflammatory conditions or idiopathically. Here, we present the case of a patient who developed LTS as a complication of granulomatosis with polyangiitis (GPA), which was misdiagnosed as asthma for 6 years. After an admission with respiratory symptoms that worsened to the extent that she required intubation, a previously well 14-year-old girl was diagnosed with GPA. Following immunosuppressive therapy, she made a good recovery and was discharged after 22 days. Over subsequent years, she developed dyspnoea and “wheeze” and a diagnosis of asthma was made. When she became pregnant, she was admitted to hospital with worsening respiratory symptoms, whereupon her “wheeze” was correctly identified as “stridor,” and subsequent investigations revealed a significant subglottic stenosis. The delay in diagnosis precluded the use of minimally invasive therapies, with the result that intermittent laser resection and open laryngotracheal reconstructive surgery were the only available treatment options. There were numerous points at which the correct diagnosis might have been made, either by proper interpretation of flow-volume loops or by calculation of the Empey or Expiratory Disproportion Indices from spirometry data

epiGBS2 : improvements and evaluation of highly multiplexed, epiGBS-based reduced representation bisulfite sequencing

Several reduced-representation bisulfite sequencing methods have been developed in recent years to determine cytosine methylation de novo in nonmodel species. Here, we present epiGBS2, a laboratory protocol based on epiGBS with a revised and user-friendly bioinformatics pipeline for a wide range of species with or without a reference genome. epiGBS2 is cost- and time-efficient and the computational workflow is designed in a user-friendly and reproducible manner. The library protocol allows a flexible choice of restriction enzymes and a double digest. The bioinformatics pipeline was integrated in the Snakemake workflow management system, which makes the pipeline easy to execute and modular, and parameter settings for important computational steps flexible. We implemented bismark for alignment and methylation analysis and we preprocessed alignment files by double masking to enable single nucleotide polymorphism calling with Freebayes (epiFreebayes). The performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and great tit (Parus major), which confirmed its overall good performance. We provide a detailed description of the laboratory protocol and an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.4764652).Publikationsfonds ML

Interventions to reduce social isolation and loneliness during COVID-19 physical distancing measures: A rapid systematic review

Background: A significant proportion of the worldwide population is at risk of social isolation and loneliness as a result of the COVID-19 pandemic. We aimed to identify effective interventions to reduce social isolation and loneliness that are compatible with COVID-19 shielding and social distancing measures. Methods and findings: In this rapid systematic review, we searched six electronic databases (Medline, Embase, Web of Science, PsycINFO, Cochrane Database of Systematic Reviews and SCOPUS) from inception to April 2020 for systematic reviews appraising interventions for loneliness and/or social isolation. Primary studies from those reviews were eligible if they included: 1) participants in a non-hospital setting; 2) interventions to reduce social isolation and/or loneliness that would be feasible during COVID-19 shielding measures; 3) a relevant control group; and 4) quantitative measures of social isolation, social support or loneliness. At least two authors independently screened studies, extracted data, and assessed risk of bias using the Downs and Black checklist. Study registration: PROSPERO CRD42020178654. We identified 45 RCTs and 13 non-randomised controlled trials; none were conducted during the COVID-19 pandemic. The nature, type, and potential effectiveness of interventions varied greatly. Effective interventions for loneliness include psychological therapies such as mindfulness, lessons on friendship, robotic pets, and social facilitation software. Few interventions improved social isolation. Overall, 37 of 58 studies were of “Fair” quality, as measured by the Downs & Black checklist. The main study limitations identified were the inclusion of studies of variable quality; the applicability of our findings to the entire population; and the current poor understanding of the types of loneliness and isolation experienced by different groups affected by the COVID-19 pandemic. Conclusions: Many effective interventions involved cognitive or educational components, or facilitated communication between peers. These interventions may require minor modifications to align with COVID-19 shielding/social distancing measures. Future high-quality randomised controlled trials conducted under shielding/social distancing constraints are urgently needed

Interventions to reduce social isolation and loneliness during COVID-19 physical distancing measures: A rapid systematic review

Background: A significant proportion of the worldwide population is at risk of social isolation and loneliness as a result of the COVID-19 pandemic. We aimed to identify effective interventions to reduce social isolation and loneliness that are compatible with COVID-19 shielding and social distancing measures. Methods and findings: In this rapid systematic review, we searched six electronic databases (Medline, Embase, Web of Science, PsycINFO, Cochrane Database of Systematic Reviews and SCOPUS) from inception to April 2020 for systematic reviews appraising interventions for loneliness and/or social isolation. Primary studies from those reviews were eligible if they included: 1) participants in a non-hospital setting; 2) interventions to reduce social isolation and/or loneliness that would be feasible during COVID-19 shielding measures; 3) a relevant control group; and 4) quantitative measures of social isolation, social support or loneliness. At least two authors independently screened studies, extracted data, and assessed risk of bias using the Downs and Black checklist. Study registration: PROSPERO CRD42020178654. We identified 45 RCTs and 13 non-randomised controlled trials; none were conducted during the COVID-19 pandemic. The nature, type, and potential effectiveness of interventions varied greatly. Effective interventions for loneliness include psychological therapies such as mindfulness, lessons on friendship, robotic pets, and social facilitation software. Few interventions improved social isolation. Overall, 37 of 58 studies were of “Fair” quality, as measured by the Downs & Black checklist. The main study limitations identified were the inclusion of studies of variable quality; the applicability of our findings to the entire population; and the current poor understanding of the types of loneliness and isolation experienced by different groups affected by the COVID-19 pandemic. Conclusions: Many effective interventions involved cognitive or educational components, or facilitated communication between peers. These interventions may require minor modifications to align with COVID-19 shielding/social distancing measures. Future high-quality randomised controlled trials conducted under shielding/social distancing constraints are urgently needed

Clinical exigencies, psychosocial realities: negotiating HIV preâ exposure prophylaxis beyond the cascade among gay, bisexual and other men who have sex with men in Canada

IntroductionNotwithstanding the efficacy of oral preâ exposure prophylaxis (PrEP) in clinical trials, a number of obstacles exist to achieving populationâ level impact among gay, bisexual and other men who have sex with men (GBM). However, few studies have explored the subjective experiences of GBM PrEP users and nonâ users in the community, outside of clinical trials. The objectives of this study were to explore GBM’s experiences of considering, accessing and using (or not using) PrEP, and to understand emerging sexual health, social and community issues among GBM in the PrEP era.MethodsFrom October 2015 to March 2016, we purposively sampled PrEPâ naïve and PrEPâ experienced GBM from community organizations and health centres in Toronto, Canada. Inâ depth, 45â to 90â minute semiâ structured interviews explored PrEP perspectives and decisionâ making, access, initiation, use over time, sexual practices and psychosocial considerations. Interviews were recorded, transcribed verbatim, uploaded into NVIVO, reviewed using thematic analysis and then contrasted with the PrEP cascade.ResultsParticipants included PrEP users (n = 15) and nonâ users (n = 14) (mean age = 36.7 years; SD = 8.2), largely gayâ identified (86.2%), cisgender male (89.7%) and white (79.3%). Themes indicate not only correspondences, but also limitations of the PrEP cascade by complicating a user/nonâ user binary and challenging the unilateral presupposition that HIV risk perception leads to PrEP acceptance. Findings further call into question assumptions of a linear stage progression and retention in care as a universal endpoint, instead revealing alternate trajectories of seasonal or intermittent PrEP use and, for some, an end goal of terminating PrEP. GBM’s narratives also revealed potent psychological/affective experiences of untethering sex from HIV anxiety; multifaceted PrEP stigma; and challenges to sexual norms and practices that complicate existing behavioural prevention strategies and sexual and social relationships.ConclusionsAn expanded PrEP cascade should consider alternate trajectories of use based on dynamic relationships and behavioural risks that may call for seasonal or intermittent use; systemic barriers in access to and sustaining PrEP; and multiple end goals including PrEP maintenance and discontinuation. Incorporating GBM’s lived experiences, evolving preferences, and psychosocial and communityâ level challenges into PrEP implementation models, rather than a circumscribed biomedical approach, may more effectively support HIV prevention and GBM’s broader sexual and psychological health.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146595/1/jia225211_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146595/2/jia225211.pd

Ixodes ricinus Tick Lipocalins: Identification, Cloning, Phylogenetic Analysis and Biochemical Characterization

BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for "Lipocalin from I. ricinus" and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50-70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4. CONCLUSIONS/SIGNIFICANCE: This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Genetic indicators of iron limitation in wild populations of \u3cem\u3eThalassiosira oceanica\u3c/em\u3e from the northeast Pacific Ocean

Assessing the iron (Fe) nutritional status of natural diatom populations has proven challenging as physiological and molecular responses can differ in diatoms of the same genus. We evaluated expression of genes encoding flavodoxin (FLDA1) and an Fe-starvation induced protein (ISIP3) as indicators of Fe limitation in the marine diatom Thalassiosira oceanica. The specificity of the response to Fe limitation was tested in cultures grown under Fe- and macronutrient-deficient conditions, as well as throughout the diurnal light cycle. Both genes showed a robust and specific response to Fe limitation in laboratory cultures and were detected in small volume samples collected from the northeast Pacific, demonstrating the sensitivity of this method. Overall, FLDA1 and ISIP3 expression was inversely related to Fe concentrations and offered insight into the Fe nutritional health of T. oceanica in the field. As T. oceanica is a species tolerant to low Fe, indications of Fe limitation in T. oceanica populations may serve as a proxy for severe Fe stress in the overall diatom community. At two shallow coastal locations, FLD1A and ISIP3 expression revealed Fe stress in areas where dissolved Fe concentrations were high, demonstrating that this approach may be powerful for identifying regions where Fe supply may not be biologically available