Integrative high-throughput study of arsenic hyper-accumulation in Pteris vittata

Arsenic is a natural contaminant in the soil and ground water, which raises considerable concerns in food safety and human health worldwide. The fernPteris vittata (Chinese brake fern) is the first identified arsenic hyperaccumulator[1]. It and its close relatives have un-paralleled ability to tolerant arsenic and feature unique arsenic metabolisms. The focus of the research presented in this thesis is to elucidate the fundamentals of arsenic tolerance and hyper-accumulation in Pteris vittata through high throughput technology and bioinformatics tools. The transcriptome of the P. vittatagametophyte under arsenate stress was obtained using RNA-Seq technology and Trinity de novo assembly. Functional annotation of the transcriptome was performed in terms of blast search, Gene Ontology term assignment, Eukaryotic Orthologous Groups (KOG) classification, and pathway analysis. Differentially expressed genes induced by arsenic stress were identified, which revealed several key players in arsenic hyper-accumulation. As part of the efforts to annotate differentially expressed genes, literature of plant arsenic tolerance was collected and built into a searchable database using the Textpresso text-mining tool, which greatly facilitates the retrieval of biological facts involving arsenic related gene. In addition, an SVM-based named-entity recognition system was constructed to identify new references to genes in literature. The results provide excellent sequence resources for arsenic tolerance study in P.vittata, and establish a platform for integrative study using data of multiple types

A Multiple 'Omics' Approach to Study the Interaction between the Vitis Vinifera Transcriptome and Epigenome and the Barossa Valley Terroir

The local growing conditions have a significant impact in grapevine (Vitis vinifera) phenotype of as well as in its epigenome. Little is known about how grapevine responds to the local environment through epigenetic modifications and any effect on gene expression in field-grown plants. Here, we characterized the leaf methylome and transcriptome of 198 Shiraz vines cultivated across twenty-two vineyards, covering six sub-regions of the Barossa Wine Zone in Australia. Samples clustered for both DNA methylation and gene expression depending on the sub-region of their origin. In addition, we identified genes with DNA methylation and expression significantly associated to measured environmental variables. Planting year and annual rainfall were the variables showing a higher influence both on DNA methylation and gene expression. Plants use long-distance signaling mechanisms to coordinate developmental and environmental cues between organs and tissues. Studies have begun to unravel the molecular nature of systemic signaling and RNA transcripts have emerged as key players by acting as mobile messengers. Along with DNA methylation and chromatin modifications, non-coding RNAs are a molecular mechanism for the epigenetic regulation of gene expression. Recent evidence in model plants suggests that dicistronic tRNA-like structures can act as mobile signals for mRNA transcripts to move between distant tissues, potentially acting as signals for development or changes in environmental conditions. However, it is not clear the extent to which dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors that contribute to their expression. We identified and analysed the expression of tRNA-mRNA dicistronic transcripts in leaves and berries from the twenty-two vineyards, using a novel pipeline developed to identify dicistronic transcripts from high-throughput short-read RNA sequencing data. Of the 124 tRNA genes that were expressed in both tissues, we detected 18 tRNA genes forming part of 19 dicistronic tRNA-mRNA molecules. We found that the presence and abundance of dicistronic molecules was tissue specific, and that each the sub-region displayed a distinct expression profile for dicistronic tRNA-mRNA transcripts. In this work we propose that DNA methylation can reflect the local growing conditions in grapevine, as a mechanism of response to its environment. We also detected the presence of dicistronic tRNA-mRNAs, which might act as signaling molecules between distant tissues. Both responses to the growing environment are especially relevant in long-lived woody plants, potentially giving them the necessary plasticity to adapt to local conditions. As grapevine is also an economically important fruit crop, understanding its response to the environment could give us a better inside of the possible mechanisms behind the expression of terroir in grapevine.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 202

Methods for Epigenetic Analyses from Long-Read Sequencing Data

Epigenetics, particularly the study of DNA methylation, is a cornerstone field for our understanding of human development and disease. DNA methylation has been included in the "hallmarks of cancer" due to its important function as a biomarker and its contribution to carcinogenesis and cancer cell plasticity. Long-read sequencing technologies, such as the Oxford Nanopore Technologies platform, have evolved the study of structural variations, while at the same time allowing direct measurement of DNA methylation on the same reads. With this, new avenues of analysis have opened up, such as long-range allele-specific methylation analysis, methylation analysis on structural variations, or relating nearby epigenetic modalities on the same read to another. Basecalling and methylation calling of Nanopore reads is a computationally expensive task which requires complex machine learning architectures. Read-level methylation calls require different approaches to data management and analysis than ones developed for methylation frequencies measured from short-read technologies or array data. The 2-dimensional nature of read and genome associated DNA methylation calls, including methylation caller uncertainties, are much more storage costly than 1-dimensional methylation frequencies. Methods for storage, retrieval, and analysis of such data therefore require careful consideration. Downstream analysis tasks, such as methylation segmentation or differential methylation calling, have the potential of benefiting from read information and allow uncertainty propagation. These avenues had not been considered in existing tools. In my work, I explored the potential of long-read DNA methylation analysis and tackled some of the challenges of data management and downstream analysis using state of the art software architecture and machine learning methods. I defined a storage standard for reference anchored and read assigned DNA methylation calls, including methylation calling uncertainties and read annotations such as haplotype or sample information. This storage container is defined as a schema for the hierarchical data format version 5, includes an index for rapid access to genomic coordinates, and is optimized for parallel computing with even load balancing. It further includes a python API for creation, modification, and data access, including convenience functions for the extraction of important quality statistics via a command line interface. Furthermore, I developed software solutions for the segmentation and differential methylation testing of DNA methylation calls from Nanopore sequencing. This implementation takes advantage of the performance benefits provided by my high performance storage container. It includes a Bayesian methylome segmentation algorithm which allows for the consensus instance segmentation of multiple sample and/or haplotype assigned DNA methylation profiles, while considering methylation calling uncertainties. Based on this segmentation, the software can then perform differential methylation testing and provides a large number of options for statistical testing and multiple testing correction. I benchmarked all tools on both simulated and publicly available real data, and show the performance benefits compared to previously existing and concurrently developed solutions. Next, I applied the methods to a cancer study on a chromothriptic cancer sample from a patient with Sonic Hedgehog Medulloblastoma. I here report regulatory genomic regions differentially methylated before and after treatment, allele-specific methylation in the tumor, as well as methylation on chromothriptic structures. Finally, I developed specialized methylation callers for the combined DNA methylation profiling of CpG, GpC, and context-free adenine methylation. These callers can be used to measure chromatin accessibility in a NOMe-seq like setup, showing the potential of long-read sequencing for the profiling of transcription factor co-binding. In conclusion, this thesis presents and subsequently benchmarks new algorithmic and infrastructural solutions for the analysis of DNA methylation data from long-read sequencing

Epigenomics of Cell Fate in Development and Disease

Epigenetic features at regulatory elements provide instructive cues for transcriptional regulation during development. However, the particular epigenetic alterations necessary for proper cell fate acquisition and differentiation are not well understood. This dissertation explores the epigenetic dynamics of regulatory elements during development and uses epigenome annotations to document inappropriate transcriptional regulation in disease. First, I summarize my contributions to developing a new algorithm for detecting differential DNA methylation, M&M. I report the application of the M&M algorithm to identify distinct classes of DNA methylation dynamics in surface ectoderm (SE) progenitor cells and SE-derived lineages: epigenome alterations, and differential DNA methylation in particular, that are present in progenitor cells are transmitted to daughter cells and consequently observed in differentiated cells. I exploit this property of DNA methylation to characterize DNA methylation dynamics in surface ectoderm embryonic tissue and SE-derived cells. Next, I use zebrafish to investigate the biological relevance of the classes of DNA methylation dynamics described in the SE context. In zebrafish, I use the pigment cell development system to understand the contribution of DNA methylation to a particular cell fate choice: melanocyte or iridophore cell fate. Next, I investigate the consequence of somatic mutations in primary liver cancer by utilizing epigenomic annotations of human tissues to distinguish putatively functional mutations from passenger mutations. Here I present support for the hypothesis that transcriptional regulatory instructions for heterologous cell types are co-opted by cancer cells during malignant tumorigenesis. Finally I present a review of the evolution of epigenetic regulation over regulatory elements. Altogether, this dissertation advances our understanding of epigenetic regulation in cell fate decisions by integrating functional genomics with developmental biology and cancer genetics

Applied Data Science Methods in Epitranscriptomic Bioinformatics

Chemical modifications on messenger RNA have been recently revealed by biological researchers to function as an essential layer of gene expression regulation. Molecular biologists from different laboratories have conducted more than 200 sets of high throughput sequencing experiments trying to capture the types and locations of messenger RNA modifications across multiple cell types and species. However, until this date, the field still lacks a bioinformatics pipeline to quantify and analyze the epitranscriptomic HTS data generated from different laboratories consistently. The thesis aims to provide an overview of questions and challenges arisen in the field of mRNA modification computational analysis. Subsequently, we will present a set of practical computational strategies for data explorations, genomic data mining, modification level quantifications, and technical artifact corrections from a data science perspective. The first chapter of the thesis provides an in-depth data exploration and visualization of m5C mRNA modification from bisulfite sequencing data. In the second chapter, we document the database construction and data consistency exploration for the transcriptomic targets of the mRNA modification related protein regulators. Besides, the second chapter presents a methodological framework for the computational representation of the domain knowledge related to the transcriptomic topology of epitranscriptomic modification. The final section of the thesis discusses the dominant technical biases existed in MeRIP-Seq, the most widely applied type of HTS data in epitranscriptomics, and it follows with a practical computational pipeline to overcome the technical error

Evolution of DNA methylation across Metazoa

DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as D. melanogaster and C. elegans. In this thesis we report on a comprehensive study of the pres- ence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. We observe that loss of individual DNMTs independently occured multiple times across ecdysozoan phyla. In several cases, this resulted in a loss of DNA methylation. In vertebrates, however, there is no single species known which lost DNA methylation. Actually, DNA methylation was greatly expanded after the 1R/2R whole genome duplication (WGD) and became a genome-wide phe- nomena. In our study of vertebrates we are not looking for losses of DNA methyltransferases and DNA methylation but are rather interested in the gain of additional DNA methyltransferase genes. In vertebrates there were a number of WGD. Most vertebrates only underwent two WGD but in the teleost lineage a third round of WGD occured and in some groups, e.g. Salmoniformes and some Cypriniformes even a forth WGD occured. The Carp-specific WGD (4R) is one of the most recent vertebrate WGD and is estimated to have occured 12.4 mya. We performed the most comprehen- sive analysis of the evolution of DNA methyltransferases after vertebrate whole-genome duplications (WGD) so far. We were able to show that the conservation of duplicated DNMT3 genes in Salmoniformes is more diverse than previously believed. We were also able to identify DNA methyltrans- ferases in Cypriniformes which have, due to their recent WGD, quite com- plex genomes. Our results show that the patterns of retained and lost DNA methyltransferases after a forth round of WGD differ between Cypriniformes and Salmoniformes. We also proposed a new nomenclature for teleost DNMT genes which correctly represents the orthology of DNMT genes for all teleost species. Next to these purely computational projects we collaborated with the Aluru lab to investigate the effects of different disturbances on zebrafish DNA methylation. One disturbance is the inactivation of DNMT3aa and DNMT3ab as single knockouts as well as a double knockout. This was the first double knockout of DNMT genes in zebrafish which was ever generated. It allows us to study the subfunctionalization of the two DNMT3a genes their effect on genome-wide DNA methylation. Given our results we hypothesize that DNMT3aa and DNMT3ab can compensate for each other to a high de- gree. DNMT3a genes have likely been subfuntionalized but their loss can be compensated by DNMT3b genes. This compensation by DNMT3b genes works well enough that no notable phenotype can be observed in double knockout zebrafish but a difference is notable on the epigenome level. The second disturbance we studied is the exposure of zebrafish to the toxic chemi- cal PCB126. We detected a moderate level of DNA methylation changes and a much larger effect on gene expression. Similar to previous reports we find little correlation between DNA methylation and gene expression changes. Therefore, while PCB126 exposure has a negative effect on DNA methyla- tion it is likely that other gene regulatory mechanisms play a role as well, possibly even a greater one. How do genes evolve and how are genes regulated are two of the main questions of modern molecular biology. In this thesis we have tried to shed more light on both questions. we have broadly expanded the phylogenetic range of species with a manually curated set of DNA methyltransferases. We have done this for ecdysozoan species which have lost all DNA methylating enzymes as well as for teleost fish which acquired more than ten copies of the, originally, two genes. We were also able to generate new insight into the subfunctionalization of the DNA methylation machinery in zebrafish and how it reacts to environmental effects.:1 Introduction 1.1 Biological introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2 Detecting DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Evolution of DNA methylation across Ecdysozoa 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3 Evolution of DNA methyltransferases after vertebrate whole genome duplications 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 The effect of DNMT3aa and DNMT3ab knockout on DNA methyla- tion in zebrafish 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish exposed to Pentachlorobiphenyl 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 6 Conclusions 6.1 Evolution of DNA methylation across Ecdysozoa . . . . . . . . . . . . . 95 6.2 Evolution of DNA methyltransferases after vertebrate whole genome duplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 6.3 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish (Danio rerio) exposed to Pentachlorobiphenyl (PCB 126). . . 107 6.4 Knockout of DNMT3aa and DNMT3ab in zebrafish (Danio rerio) . . . . . . 108 Bibliography 11

Genomic Regulation Of Abiotic Stress Response In The Soil Nematode Oscheius Tipulae

Oscheius tipulae is a species of free-living soil nematode that can be found in ecosystems worldwide. Because of this, individuals must be able to respond to heat, freezing, and desiccation stresses in order to survive. They do this by producing a suite of cellular responses, some of which are necessary to survive multiple stresses, and some are stress-specific. While these cellular responses are well known, the ways in which they are regulated in a genome-wide context are not. In this project, multiple high throughput sequencing and bioinformatics analyses were utilized to answer this question. First, the O. tipulae genome was sequenced via Illumina HiSeq, assembled, and annotated. An RNA-Seq experiment was performed to determine transcription patterns within stress responses. Pooled nematode samples were subjected to heat, freezing, or desiccation stress prior to RNA sequencing and read mapping. Results showed that shared cellular responses were controlled by the upregulation of both shared and stress-specific genes. This suggests that the genome remains efficient by utilizing overlapping response genes and reinforcing them with stress-specific genes. Whole genome bisulfite sequencing and MethylCap-Seq analyses were performed to assess DNA cytosine methylation presence in O. tipulae and the model organism Caenorhabditis elegans and to determine its role in the abiotic stress response process in O. tipulae. Methylated cytosines were found in both O. tipulae and C. elegans, contradicting the historical belief that cytosine methylation is absent in nematodes. Changes in DNA methylation were not associated with the abiotic stress response as very few methylation cites were found within upregulated genes. This project utilized new sequencing technologies and various bioinformatics programs to provide an in-depth look into the genome-wide responses to abiotic stress in O. tipulae

Inter-individual variation of the human epigenome & applications

Genome-wide association studies (GWAS) have led to the discovery of genetic variants influencing human phenotypes in health and disease. However, almost two decades later, most human traits can still not be accurately predicted from common genetic variants. Moreover, genetic variants discovered via GWAS mostly map to the non-coding genome and have historically resisted interpretation via mechanistic models. Alternatively, the epigenome lies in the cross-roads between genetics and the environment. Thus, there is great excitement towards the mapping of epigenetic inter-individual variation since its study may link environmental factors to human traits that remain unexplained by genetic variants. For instance, the environmental component of the epigenome may serve as a source of biomarkers for accurate, robust and interpretable phenotypic prediction on low-heritability traits that cannot be attained by classical genetic-based models. Additionally, its research may provide mechanisms of action for genetic associations at non-coding regions that mediate their effect via the epigenome. The aim of this thesis was to explore epigenetic inter-individual variation and to mitigate some of the methodological limitations faced towards its future valorisation.Chapter 1 is dedicated to the scope and aims of the thesis. It begins by describing historical milestones and basic concepts in human genetics, statistical genetics, the heritability problem and polygenic risk scores. It then moves towards epigenetics, covering the several dimensions it encompasses. It subsequently focuses on DNA methylation with topics like mitotic stability, epigenetic reprogramming, X-inactivation or imprinting. This is followed by concepts from epigenetic epidemiology such as epigenome-wide association studies (EWAS), epigenetic clocks, Mendelian randomization, methylation risk scores and methylation quantitative trait loci (mQTL). The chapter ends by introducing the aims of the thesis.Chapter 2 focuses on stochastic epigenetic inter-individual variation resulting from processes occurring post-twinning, during embryonic development and early life. Specifically, it describes the discovery and characterisation of hundreds of variably methylated CpGs in the blood of healthy adolescent monozygotic (MZ) twins showing equivalent variation among co-twins and unrelated individuals (evCpGs) that could not be explained only by measurement error on the DNA methylation microarray. DNA methylation levels at evCpGs were shown to be stable short-term but susceptible to aging and epigenetic drift in the long-term. The identified sites were significantly enriched at the clustered protocadherin loci, known for stochastic methylation in neurons in the context of embryonic neurodevelopment. Critically, evCpGs were capable of clustering technical and longitudinal replicates while differentiating young MZ twins. Thus, discovered evCpGs can be considered as a first prototype towards universal epigenetic fingerprint, relevant in the discrimination of MZ twins for forensic purposes, currently impossible with standard DNA profiling. Besides, DNA methylation microarrays are the preferred technology for EWAS and mQTL mapping studies. However, their probe design inherently assumes that the assayed genomic DNA is identical to the reference genome, leading to genetic artifacts whenever this assumption is not fulfilled. Building upon the previous experience analysing microarray data, Chapter 3 covers the development and benchmarking of UMtools, an R-package for the quantification and qualification of genetic artifacts on DNA methylation microarrays based on the unprocessed fluorescence intensity signals. These tools were used to assemble an atlas on genetic artifacts encountered on DNA methylation microarrays, including interactions between artifacts or with X-inactivation, imprinting and tissue-specific regulation. Additionally, to distinguish artifacts from genuine epigenetic variation, a co-methylation-based approach was proposed. Overall, this study revealed that genetic artifacts continue to filter through into the reported literature since current methodologies to address them have overlooked this challenge.Furthermore, EWAS, mQTL and allele-specific methylation (ASM) mapping studies have all been employed to map epigenetic variation but require matching phenotypic/genotypic data and can only map specific components of epigenetic inter-individual variation. Inspired by the previously proposed co-methylation strategy, Chapter 4 describes a novel method to simultaneously map inter-haplotype, inter-cell and inter-individual variation without these requirements. Specifically, binomial likelihood function-based bootstrap hypothesis test for co-methylation within reads (Binokulars) is a randomization test that can identify jointly regulated CpGs (JRCs) from pooled whole genome bisulfite sequencing (WGBS) data by solely relying on joint DNA methylation information available in reads spanning multiple CpGs. Binokulars was tested on pooled WGBS data in whole blood, sperm and combined, and benchmarked against EWAS and ASM. Our comparisons revealed that Binokulars can integrate a wide range of epigenetic phenomena under the same umbrella since it simultaneously discovered regions associated with imprinting, cell type- and tissue-specific regulation, mQTL, ageing or even unknown epigenetic processes. Finally, we verified examples of mQTL and polymorphic imprinting by employing another novel tool, JRC_sorter, to classify regions based on epigenotype models and non-pooled WGBS data in cord blood. In the future, we envision how this cost-effective approach can be applied on larger pools to simultaneously highlight regions of interest in the methylome, a highly relevant task in the light of the post-GWAS era.Moving towards future applications of epigenetic inter-individual variation, Chapters 5 and 6 are dedicated to solving some of methodological issues faced in translational epigenomics.Firstly, due to its simplicity and well-known properties, linear regression is the starting point methodology when performing prediction of a continuous outcome given a set of predictors. However, linear regression is incompatible with missing data, a common phenomenon and a huge threat to the integrity of data analysis in empirical sciences, including (epi)genomics. Chapter 5 describes the development of combinatorial linear models (cmb-lm), an imputation-free, CPU/RAM-efficient and privacy-preserving statistical method for linear regression prediction on datasets with missing values. Cmb-lm provide prediction errors that take into account the pattern of missing values in the incomplete data, even at extreme missingness. As a proof-of-concept, we tested cmb-lm in the context of epigenetic ageing clocks, one of the most popular applications of epigenetic inter-individual variation. Overall, cmb-lm offer a simple and flexible methodology with a wide range of applications that can provide a smooth transition towards the valorisation of linear models in the real world, where missing data is almost inevitable. Beyond microarrays, due to its high accuracy, reliability and sample multiplexing capabilities, massively parallel sequencing (MPS) is currently the preferred methodology of choice to translate prediction models for traits of interests into practice. At the same time, tobacco smoking is a frequent habit sustained by more than 1.3 billion people in 2020 and a leading (and preventable) health risk factor in the modern world. Predicting smoking habits from a persistent biomarker, such as DNA methylation, is not only relevant to account for self-reporting bias in public health and personalized medicine studies, but may also allow broadening forensic DNA phenotyping. Previously, a model to predict whether someone is a current, former, or never smoker had been published based on solely 13 CpGs from the hundreds of thousands included in the DNA methylation microarray. However, a matching lab tool with lower marker throughput, and higher accuracy and sensitivity was missing towards translating the model in practice. Chapter 6 describes the development of an MPS assay and data analysis pipeline to quantify DNA methylation on these 13 smoking-associated biomarkers for the prediction of smoking status. Though our systematic evaluation on DNA standards of known methylation levels revealed marker-specific amplification bias, our novel tool was still able to provide highly accurate and reproducible DNA methylation quantification and smoking habit prediction. Overall, our MPS assay allows the technological transfer of DNA methylation microarray findings and models to practical settings, one step closer towards future applications.Finally, Chapter 7 provides a general discussion on the results and topics discussed across Chapters 2-6. It begins by summarizing the main findings across the thesis, including proposals for follow-up studies. It then covers technical limitations pertaining bisulfite conversion and DNA methylation microarrays, but also more general considerations such as restricted data access. This chapter ends by covering the outlook of this PhD thesis, including topics such as bisulfite-free methods, third-generation sequencing, single-cell methylomics, multi-omics and systems biology.<br/