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

Atas das Oitavas Jornadas de Informática da Universidade de Évora

Atas das Oitavas Jornadas de Informática da Universidade de Évora realizadas em Março de 2018

Tuning the Computational Effort: An Adaptive Accuracy-aware Approach Across System Layers

This thesis introduces a novel methodology to realize accuracy-aware systems, which will help designers integrate accuracy awareness into their systems. It proposes an adaptive accuracy-aware approach across system layers that addresses current challenges in that domain, combining and tuning accuracy-aware methods on different system layers. To widen the scope of accuracy-aware computing including approximate computing for other domains, this thesis presents innovative accuracy-aware methods and techniques for different system layers. The required tuning of the accuracy-aware methods is integrated into a configuration layer that tunes the available knobs of the accuracy-aware methods integrated into a system

Secure Computation Protocols for Privacy-Preserving Machine Learning

Machine Learning (ML) profitiert erheblich von der Verfügbarkeit großer Mengen an Trainingsdaten, sowohl im Bezug auf die Anzahl an Datenpunkten, als auch auf die Anzahl an Features pro Datenpunkt. Es ist allerdings oft weder möglich, noch gewollt, mehr Daten unter zentraler Kontrolle zu aggregieren. Multi-Party-Computation (MPC)-Protokolle stellen eine Lösung dieses Dilemmas in Aussicht, indem sie es mehreren Parteien erlauben, ML-Modelle auf der Gesamtheit ihrer Daten zu trainieren, ohne die Eingabedaten preiszugeben. Generische MPC-Ansätze bringen allerdings erheblichen Mehraufwand in der Kommunikations- und Laufzeitkomplexität mit sich, wodurch sie sich nur beschränkt für den Einsatz in der Praxis eignen. Das Ziel dieser Arbeit ist es, Privatsphäreerhaltendes Machine Learning mittels MPC praxistauglich zu machen. Zuerst fokussieren wir uns auf zwei Anwendungen, lineare Regression und Klassifikation von Dokumenten. Hier zeigen wir, dass sich der Kommunikations- und Rechenaufwand erheblich reduzieren lässt, indem die aufwändigsten Teile der Berechnung durch Sub-Protokolle ersetzt werden, welche auf die Zusammensetzung der Parteien, die Verteilung der Daten, und die Zahlendarstellung zugeschnitten sind. Insbesondere das Ausnutzen dünnbesetzter Datenrepräsentationen kann die Effizienz der Protokolle deutlich verbessern. Diese Beobachtung verallgemeinern wir anschließend durch die Entwicklung einer Datenstruktur für solch dünnbesetzte Daten, sowie dazugehöriger Zugriffsprotokolle. Aufbauend auf dieser Datenstruktur implementieren wir verschiedene Operationen der Linearen Algebra, welche in einer Vielzahl von Anwendungen genutzt werden. Insgesamt zeigt die vorliegende Arbeit, dass MPC ein vielversprechendes Werkzeug auf dem Weg zu Privatsphäre-erhaltendem Machine Learning ist, und die von uns entwickelten Protokolle stellen einen wesentlichen Schritt in diese Richtung dar.Machine learning (ML) greatly benefits from the availability of large amounts of training data, both in terms of the number of samples, and the number of features per sample. However, aggregating more data under centralized control is not always possible, nor desirable, due to security and privacy concerns, regulation, or competition. Secure multi-party computation (MPC) protocols promise a solution to this dilemma, allowing multiple parties to train ML models on their joint datasets while provably preserving the confidentiality of the inputs. However, generic approaches to MPC result in large computation and communication overheads, which limits the applicability in practice. The goal of this thesis is to make privacy-preserving machine learning with secure computation practical. First, we focus on two high-level applications, linear regression and document classification. We show that communication and computation overhead can be greatly reduced by identifying the costliest parts of the computation, and replacing them with sub-protocols that are tailored to the number and arrangement of parties, the data distribution, and the number representation used. One of our main findings is that exploiting sparsity in the data representation enables considerable efficiency improvements. We go on to generalize this observation, and implement a low-level data structure for sparse data, with corresponding secure access protocols. On top of this data structure, we develop several linear algebra algorithms that can be used in a wide range of applications. Finally, we turn to improving a cryptographic primitive named vector-OLE, for which we propose a novel protocol that helps speed up a wide range of secure computation tasks, within private machine learning and beyond. Overall, our work shows that MPC indeed offers a promising avenue towards practical privacy-preserving machine learning, and the protocols we developed constitute a substantial step in that direction