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1.  Next-Generation Sequencing in the Understanding of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Biology 
Viruses  2016;8(4):92.
Non-Sanger-based novel nucleic acid sequencing techniques, referred to as Next-Generation Sequencing (NGS), provide a rapid, reliable, high-throughput, and massively parallel sequencing methodology that has improved our understanding of human cancers and cancer-related viruses. NGS has become a quintessential research tool for more effective characterization of complex viral and host genomes through its ever-expanding repertoire, which consists of whole-genome sequencing, whole-transcriptome sequencing, and whole-epigenome sequencing. These new NGS platforms provide a comprehensive and systematic genome-wide analysis of genomic sequences and a full transcriptional profile at a single nucleotide resolution. When combined, these techniques help unlock the function of novel genes and the related pathways that contribute to the overall viral pathogenesis. Ongoing research in the field of virology endeavors to identify the role of various underlying mechanisms that control the regulation of the herpesvirus biphasic lifecycle in order to discover potential therapeutic targets and treatment strategies. In this review, we have complied the most recent findings about the application of NGS in Kaposi’s sarcoma-associated herpesvirus (KSHV) biology, including identification of novel genomic features and whole-genome KSHV diversities, global gene regulatory network profiling for intricate transcriptome analyses, and surveying of epigenetic marks (DNA methylation, modified histones, and chromatin remodelers) during de novo, latent, and productive KSHV infections.
doi:10.3390/v8040092
PMCID: PMC4848587  PMID: 27043613
Kaposi’s sarcoma-associated herpesvirus; KSHV; next-generation sequencing; genomics; transcriptomics; epigenomics; virology
2.  H2A.Z Demarcates Intergenic Regions of the Plasmodium falciparum Epigenome That Are Dynamically Marked by H3K9ac and H3K4me3 
PLoS Pathogens  2010;6(12):e1001223.
Epigenetic regulatory mechanisms and their enzymes are promising targets for malaria therapeutic intervention; however, the epigenetic component of gene expression in P. falciparum is poorly understood. Dynamic or stable association of epigenetic marks with genomic features provides important clues about their function and helps to understand how histone variants/modifications are used for indexing the Plasmodium epigenome. We describe a novel, linear amplification method for next-generation sequencing (NGS) that allows unbiased analysis of the extremely AT-rich Plasmodium genome. We used this method for high resolution, genome-wide analysis of a histone H2A variant, H2A.Z and two histone H3 marks throughout parasite intraerythrocytic development. Unlike in other organisms, H2A.Z is a constant, ubiquitous feature of euchromatic intergenic regions throughout the intraerythrocytic cycle. The almost perfect colocalisation of H2A.Z with H3K9ac and H3K4me3 suggests that these marks are preferentially deposited on H2A.Z-containing nucleosomes. By performing RNA-seq on 8 time-points, we show that acetylation of H3K9 at promoter regions correlates very well with the transcriptional status whereas H3K4me3 appears to have stage-specific regulation, being low at early stages, peaking at trophozoite stage, but does not closely follow changes in gene expression. Our improved NGS library preparation procedure provides a foundation to exploit the malaria epigenome in detail. Furthermore, our findings place H2A.Z at the cradle of P. falciparum epigenetic regulation by stably defining intergenic regions and providing a platform for dynamic assembly of epigenetic and other transcription related complexes.
Author Summary
Plasmodium falciparum is a unicellular pathogen that is responsible for the most severe form of malaria. Similar to other eukaryotic organisms, its genome is organized into chromosomes by proteins called histones. Modification or replacement of these histones has marked effects on the packaging grade of DNA and instructs the recruitment of protein complexes, thereby regulating essential cellular processes such as gene expression and replication. Here we unveil the genome-wide localization of two histone H3 modifications (K9ac/K4me3) and a histone variant, H2A.Z, during development of the parasite in the human red blood cells. We find that all three epigenetic features are predominantly present in intergenic regions of the P. falciparum genome, suggesting an interconnecting role in regulation of gene expression. H2A.Z levels appear to be largely invariable throughout intraerythrocytic development while placement/removal of the histone marks is dynamic with H3K9ac and H3K4me3 being transcription-coupled and stage-specific, respectively. These observations support a model in which H2A.Z-containing nucleosomes serve to demarcate regulatory regions in the parasite's genome and promote transcription initiation by guiding chromatin modifying and transcription initiating complexes. The findings and methodological developments presented in this paper provide a cornerstone for future epigenome research in eukaryotic pathogens and vital information to understand and to interfere with parasite development and survival.
doi:10.1371/journal.ppat.1001223
PMCID: PMC3002978  PMID: 21187892
3.  Comparative epigenomics: defining and utilizing epigenomic variations across species, time-course, and individuals 
Epigenomic profiling, by revealing genome-wide distributions of epigenetic modifications, generated a large amount of structural information about the chromosomes. Epigenomic analysis has quickly become a big data science, posing tremendous challenges on its translation into knowledge. To meet this challenge, comparative analysis of epigenomes, dubbed comparative epigenomics, has emerged as an active research area. Here, we summarize the recent developments in comparative epigenomic analyses into three major directions, namely the comparisons across species, the time-course of a biological process, and individuals. We review the main ideas, methods, and findings in each direction, and discuss the implications to understanding the regulatory functions of the genomes.
Epigenomes play pivotal roles in cell identity, organismal development and disease processes [1, 2], contribute to regulating cognition and behavior [3] and reflect personal variation [4]. By integrating environmental signals with genomic instructions, epigenomes are instrumental in bridging genotypic variation and phenotypic diversity. The rapid growth of high throughput sequencing has substantially reduced the costs of mapping epigenomes. A few new common understandings have been established through epigenomic profiling. First, each cell type possesses characteristic chromatin states [5-8]. Second, cis regulatory elements possess specific chromatin signatures, characterized by combinations of epigenomic marks [9-11]. Third, a large class of new genes that produce long intergenic non-coding RNAs (lincRNAs) possess similar epigenomic characteristics to coding genes, and thus can be identified and annotated in the genome [12, 13].
Epigenomic analysis has quickly become a big data science, posing tremendous challenges on its translation into knowledge. One dimension of the humongous growth of epigenomic data is in size, which is powered by three orthogonal engines. First, high-throughput sequencing enabled gigantic scales of data generation. Second, the enhanced data federation merged data across multiple labs and multiple institutions. Third, the raw data were transformed into processed data by analysis software, which becomes a multiplier of the (raw) big data. A second dimension of the growth of epigenomic data is in complexity and heterogeneity, which is at least partially by multi-dimensional data reflecting different aspects of the epigenetic states, including but not limited to protein-DNA interactions, histone and DNA modifications, long-range interactions, RNA-chromatin interactions, and the increasingly popular time-course experimental design.
The challenge of translating epigenomic data into the knowledge of regulatory functions of the genome has been met by the recently developed “comparative epigenomics” approach [14-17]. Comparative analysis is perhaps the oldest and the most essential approach to study biology. The soon that epigenomic maps were produced in a genome-wide manner, comparative analysis between cell types were started with embryonic stem cells and adult cell types [8, 18, 19]. Such comparisons led to the early discovery that embryonic stem cells possess a specific combination of epigenomic marks, dubbed bivalent domains [20]. This intuitive cell type comparison provided early insights but did not fully reveal the information buried in epigenomic data. Since then, new comparison methods have been explored and a few breakthroughs were made in the recent years.
doi:10.1002/wsbm.1274
PMCID: PMC4851441  PMID: 25044241
4.  DNA Methylation: A Timeline of Methods and Applications 
DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. An epigenetic modification associated with gene regulation, DNA methylation is of paramount importance to biological health and disease. Recently, the quest to unravel the Human Epigenome commenced, calling for a modernization of previous DNA methylation profiling techniques. Here, we describe the major developments in the methodologies used over the past three decades to examine the elusive epigenome (or methylome). The earliest techniques were based on the separation of methylated and unmethylated cytosines via chromatography. The following years would see molecular techniques being employed to indirectly examine DNA methylation levels at both a genome-wide and locus-specific context, notably immunoprecipitation via anti-5′methylcytosine and selective digestion with methylation-sensitive restriction endonucleases. With the advent of sodium bisulfite treatment of DNA, a deamination reaction that converts cytosine to uracil only when unmethylated, the epigenetic modification can now be identified in the same manner as a DNA base-pair change. More recently, these three techniques have been applied to more technically advanced systems such as DNA microarrays and next-generation sequencing platforms, bringing us closer to unveiling a complete human epigenetic profile.
doi:10.3389/fgene.2011.00074
PMCID: PMC3268627  PMID: 22303369
DNA; methylation; bisulfite; sequencing; methods
5.  An emerging place for lung cancer genomics in 2013 
Journal of Thoracic Disease  2013;5(Suppl 5):S491-S497.
Lung cancer is a disease with a dismal prognosis and is the biggest cause of cancer deaths in many countries. Nonetheless, rapid technological developments in genome science promise more effective prevention and treatment strategies. Since the Human Genome Project, scientific advances have revolutionized the diagnosis and treatment of human cancers, including thoracic cancers. The latest, massively parallel, next generation sequencing (NGS) technologies offer much greater sequencing capacity than traditional, capillary-based Sanger sequencing. These modern but costly technologies have been applied to whole genome-, and whole exome sequencing (WGS and WES) for the discovery of mutations and polymorphisms, transcriptome sequencing for quantification of gene expression, small ribonucleic acid (RNA) sequencing for microRNA profiling, large scale analysis of deoxyribonucleic acid (DNA) methylation and chromatin immunoprecipitation mapping of DNA-protein interaction.
With the rise of personalized cancer care, based on the premise of precision medicine, sequencing technologies are constantly changing. To date, the genomic landscape of lung cancer has been captured in several WGS projects. Such work has not only contributed to our understanding of cancer biology, but has also provided impetus for technical advances that may improve our ability to accurately capture the cancer genome. Issues such as short read lengths contribute to sequenced libraries that contain challenging gaps in the aligned genome. Emerging platforms promise longer reads as well as the ability to capture a range of epigenomic signals. In addition, ongoing optimization of bioinformatics strategies for data analysis and interpretation are critical, especially for the differentiation between driver and passenger mutations.
Moreover, broader deployment of these and future generations of platforms, coupled with an increasing bioinformatics workforce with access to highly sophisticated technologies, could see many of these discoveries translated to the clinic at a rapid pace. We look forward to these advances making a difference for the many patients we treat in the Asia-Pacific region and around the world.
doi:10.3978/j.issn.2072-1439.2013.10.06
PMCID: PMC3804884  PMID: 24163742
High-throughput nucleotide sequencing; genomics; lung neoplasms; non-small cell lung carcinoma (NSCLC); small cell lung carcinoma (SCLC)
6.  Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots 
Clinical Epigenetics  2016;8:81.
Background
In utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific disease-relevant biomarkers may prove challenging. This has led to an increased interest in epigenome-wide association studies using dried blood spots (DBS) routinely collected in perinatal screening programs. Such programs are in place in numerous countries around the world producing large and unique biobanks. However, availability of this biological material is highly limited as each DBS is made only from a few droplets of blood and storage conditions may be suboptimal for epigenetic studies. Furthermore, as relevant markers may reside outside gene bodies, epigenome-wide interrogation is needed.
Results
Here we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60 ng DNA) from filter cards archived for up to 16 years. The enrichment profile, sequence quality and distribution of reads across genetic regions were comparable between samples archived 16 years, 4 years and a freshly prepared control sample.
Conclusions
In summary, we show that high-quality MeDIP-seq data is achievable from neonatal screening filter cards stored at room temperature, thereby providing information on annotated as well as on non-RefSeq genes and repetitive elements. Moreover, the quantity of DNA from one DBS punch proved sufficient allowing for multiple epigenome studies using one single DBS.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-016-0242-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13148-016-0242-1
PMCID: PMC4960904  PMID: 27462375
DNA methylation; Archival dried blood spots; MeDIP-seq; Low input; Genome-wide
7.  Epigenome remodelling in breast cancer: insights from an early in vitro model of carcinogenesis 
Epigenetic gene regulation has influence over a diverse range of cellular functions, including the maintenance of pluripotency, differentiation, and cellular identity, and is deregulated in many diseases, including cancer. Whereas the involvement of epigenetic dysregulation in cancer is well documented, much of the mechanistic detail involved in triggering these changes remains unclear. In the current age of genomics, the development of new sequencing technologies has seen an influx of genomic and epigenomic data and drastic improvements in both resolution and coverage. Studies in cancer cell lines and clinical samples using next-generation sequencing are rapidly delivering spectacular insights into the nature of the cancer genome and epigenome. Despite these improvements in technology, the timing and relationship between genetic and epigenetic changes that occur during the process of carcinogenesis are still unclear. In particular, what changes to the epigenome are playing a driving role during carcinogenesis and what influence the temporal nature of these changes has on cancer progression are not known. Understanding the early epigenetic changes driving breast cancer has the exciting potential to provide a novel set of therapeutic targets or early-disease biomarkers or both. Therefore, it is important to find novel systems that permit the study of initial epigenetic events that potentially occur during the first stages of breast cancer. Non-malignant human mammary epithelial cells (HMECs) provide an exciting in vitro model of very early breast carcinogenesis. When grown in culture, HMECs are able to temporarily escape senescence and acquire a pre-malignant breast cancer-like phenotype (variant HMECs, or vHMECs). Cultured HMECs are composed mainly of cells from the basal breast epithelial layer. Therefore, vHMECs are considered to represent the basal-like subtype of breast cancer. The transition from HMECs to vHMECs in culture recapitulates the epigenomic phenomena that occur during the progression from normal breast to pre-malignancy. Therefore, the HMEC model system provides the unique opportunity to study the very earliest epigenomic aberrations occurring during breast carcinogenesis and can give insight into the sequence of epigenomic events that lead to breast malignancy. This review provides an overview of epigenomic research in breast cancer and discusses in detail the utility of the HMEC model system to discover early epigenomic changes involved in breast carcinogenesis.
doi:10.1186/bcr3237
PMCID: PMC4053120  PMID: 23168266
8.  PD_NGSAtlas: a reference database combining next-generation sequencing epigenomic and transcriptomic data for psychiatric disorders 
BMC Medical Genomics  2014;7:71.
Background
Psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BP) are projected to lead the global disease burden within the next decade. Several lines of evidence suggest that epigenetic- or genetic-mediated dysfunction is frequently present in these disorders. To date, the inheritance patterns have been complicated by the problem of integrating epigenomic and transcriptomic factors that have yet to be elucidated. Therefore, there is a need to build a comprehensive database for storing epigenomic and transcriptomic data relating to psychiatric disorders.
Description
We have developed the PD_NGSAtlas, which focuses on the efficient storage of epigenomic and transcriptomic data based on next-generation sequencing and on the quantitative analyses of epigenetic and transcriptional alterations involved in psychiatric disorders. The current release of the PD_NGSAtlas contains 43 DNA methylation profiles and 37 transcription profiles detected by MeDIP-Seq and RNA-Seq, respectively, in two distinct brain regions and peripheral blood of SZ, BP and non-psychiatric controls. In addition to these data that were generated in-house, we have included, and will continue to include, published DNA methylation and gene expression data from other research groups, with a focus on psychiatric disorders. A flexible query engine has been developed for the acquisition of methylation profiles and transcription profiles for special genes or genomic regions of interest of the selected samples. Furthermore, the PD_NGSAtlas offers online tools for identifying aberrantly methylated and expressed events involved in psychiatric disorders. A genome browser has been developed to provide integrative and detailed views of multidimensional data in a given genomic context, which can help researchers understand molecular mechanisms from epigenetic and transcriptional perspectives. Moreover, users can download the methylation and transcription data for further analyses.
Conclusions
The PD_NGSAtlas aims to provide storage of epigenomic and transcriptomic data as well as quantitative analyses of epigenetic and transcriptional alterations involved in psychiatric disorders. The PD_NGSAtlas will be a valuable data resource and will enable researchers to investigate the pathophysiology and aetiology of disease in detail. The database is available at http://bioinfo.hrbmu.edu.cn/pd_ngsatlas/.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-014-0071-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12920-014-0071-z
PMCID: PMC4308070  PMID: 25551368
Schizophrenia; Bipolar disorder; Next-generation sequencing; Epigenomic and transcriptomic data; Brain; Blood
9.  Next-Generation Sequencing and Epigenomics Research: A Hammer in Search of Nails 
Genomics & Informatics  2014;12(1):2-11.
After the initial enthusiasm of the human genome project, it became clear that without additional data pertaining to the epigenome, i.e., how the genome is marked at specific developmental periods, in different tissues, as well as across individuals and species-the promise of the genome sequencing project in understanding biology cannot be fulfilled. This realization prompted several large-scale efforts to map the epigenome, most notably the Encyclopedia of DNA Elements (ENCODE) project. While there is essentially a single genome in an individual, there are hundreds of epigenomes, corresponding to various types of epigenomic marks at different developmental times and in multiple tissue types. Unprecedented advances in next-generation sequencing (NGS) technologies, by virtue of low cost and high speeds that continue to improve at a rate beyond what is anticipated by Moore's law for computer hardware technologies, have revolutionized molecular biology and genetics research, and have in turn prompted innovative ways to reduce the problem of measuring cellular events involving DNA or RNA into a sequencing problem. In this article, we provide a brief overview of the epigenome, the various types of epigenomic data afforded by NGS, and some of the novel discoveries yielded by the epigenomics projects. We also provide ample references for the reader to get in-depth information on these topics.
doi:10.5808/GI.2014.12.1.2
PMCID: PMC3990762  PMID: 24748856
chromatin accessibility; epigenomics; methylation; next-generation sequencing; regulation
10.  Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions 
Ageing research reviews  2009;9(3):315-323.
Recent technological advances that allow faster and cheaper DNA sequencing are now driving biological and medical research. In this review, we provide an overview of state-of-the-art next-generation sequencing (NGS) platforms and their applications, including in genome sequencing and resequencing, transcriptional profiling (RNA-Seq) and high-throughput survey of DNA-protein interactions (ChIP-Seq) and of the epigenome. Particularly, we focus on how new methods made possible by NGS can help unravel the biological and genetic mechanisms of aging, longevity and age-related diseases. In the same way, however, NGS platforms open discovery not available before, they also give rise to new challenges, in particular in processing, analyzing and interpreting the data. Bioinformatics and software issues plus statistical difficulties in genome-wide studies are discussed, as well as the use of targeted sequencing to decrease costs and facilitate statistical analyses. Lastly, we discuss a number of methods to gather biological insights from massive amounts of data, such as functional enrichment, transcriptional regulation and network analyses. Although in the fast-moving field of NGS new platforms will soon take center stage, the approaches made possible by NGS will be at the basis of molecular biology, genetics and systems biology for years to come, making them instrumental for research on aging.
doi:10.1016/j.arr.2009.10.006
PMCID: PMC2878865  PMID: 19900591
bioinformatics; epigenetics; functional genomics; senescence; systems biology
11.  Diabetic embryopathy: A role for the epigenome? 
Embryonic development under adverse conditions, such as maternal diabetes or obesity during pregnancy, constitutes a major risk factor for birth defects, as well as for long-term health consequences and disease susceptibility in the offspring. While contributions from epigenetic changes have been invoked previously to explain the long-term changes in terms of developmental programming, we here review how maternal metabolism may directly affect the embryonic epigenome in relationship to teratogenic processes. We consider four epigenetic modalities – DNA methylation, non-coding RNA, transcription factors, and histone modifications – and their contribution to epigenetic memory, and discuss how epigenomic changes may mediate the altered control of embryonic gene expression brought about by maternal diabetes. In combination, the epigenomic modalities serve to define transcription-permissive domains of the genome, resulting in distinct epigenomic landscapes in different developmental cell types. We evaluate experimental approaches to characterize the epigenome in adverse pregnancy conditions, highlighting the role of next-generation sequencing on the technological side, while emphasizing the necessity to study defined cell populations in terms of biologic impact. Finally, we outline the challenges in moving from findings that correlate epigenomics to developmental phenotypes to scenarios that establish teratogenic causality.
doi:10.1002/bdra.20807
PMCID: PMC3152694  PMID: 21538816
12.  Next Generation Sequencing: Advances in Characterizing the Methylome  
Genes  2010;1(2):143-165.
Epigenetic modifications play an important role in lymphoid malignancies. This has been evidenced by the large body of work published using microarray technologies to generate methylation profiles for numerous types and subtypes of lymphoma and leukemia. These studies have shown the importance of defining the epigenome so that we can better understand the biology of lymphoma. Recent advances in DNA sequencing technology have transformed the landscape of epigenomic analysis as we now have the ability to characterize the genome-wide distribution of chromatin modifications and DNA methylation using next-generation sequencing. To take full advantage of the throughput of next-generation sequencing, there are many methodologies that have been developed and many more that are currently being developed. Choosing the appropriate methodology is fundamental to the outcome of next-generation sequencing studies. In this review, published technologies and methodologies applicable to studying the methylome are presented. In addition, progress towards defining the methylome in lymphoma is discussed and prospective directions that have been made possible as a result of next-generation sequencing technology. Finally, methodologies are introduced that have not yet been published but that are being explored in the pursuit of defining the lymphoma methylome.
doi:10.3390/genes1010143
PMCID: PMC3954092  PMID: 24710039
lymphoma; leukemia; next-generation sequencing; methylation; epigenome
13.  Whole genome sequencing for lung cancer 
Journal of Thoracic Disease  2012;4(2):155-163.
Lung cancer is a leading cause of cancer related morbidity and mortality globally, and carries a dismal prognosis. Improved understanding of the biology of cancer is required to improve patient outcomes. Next-generation sequencing (NGS) is a powerful tool for whole genome characterisation, enabling comprehensive examination of somatic mutations that drive oncogenesis. Most NGS methods are based on polymerase chain reaction (PCR) amplification of platform-specific DNA fragment libraries, which are then sequenced. These techniques are well suited to high-throughput sequencing and are able to detect the full spectrum of genomic changes present in cancer. However, they require considerable investments in time, laboratory infrastructure, computational analysis and bioinformatic support. Next-generation sequencing has been applied to studies of the whole genome, exome, transcriptome and epigenome, and is changing the paradigm of lung cancer research and patient care. The results of this new technology will transform current knowledge of oncogenic pathways and provide molecular targets of use in the diagnosis and treatment of cancer. Somatic mutations in lung cancer have already been identified by NGS, and large scale genomic studies are underway. Personalised treatment strategies will improve care for those likely to benefit from available therapies, while sparing others the expense and morbidity of futile intervention. Organisational, computational and bioinformatic challenges of NGS are driving technological advances as well as raising ethical issues relating to informed consent and data release. Differentiation between driver and passenger mutations requires careful interpretation of sequencing data. Challenges in the interpretation of results arise from the types of specimens used for DNA extraction, sample processing techniques and tumour content. Tumour heterogeneity can reduce power to detect mutations implicated in oncogenesis. Next-generation sequencing will facilitate investigation of the biological and clinical implications of such variation. These techniques can now be applied to single cells and free circulating DNA, and possibly in the future to DNA obtained from body fluids and from subpopulations of tumour. As costs reduce, and speed and processing accuracy increase, NGS technology will become increasingly accessible to researchers and clinicians, with the ultimate goal of improving the care of patients with lung cancer.
doi:10.3978/j.issn.2072-1439.2012.02.01
PMCID: PMC3378223  PMID: 22833821
High-throughput nucleotide sequencing; DNA sequence analysis; lung neoplasms; non-small cell lung carcinoma; small cell lung carcinoma
14.  TELP, a sensitive and versatile library construction method for next-generation sequencing 
Nucleic Acids Research  2014;43(6):e35.
Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (i) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (ii) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (iii) achieved paired-end sequencing of the ChIP-seq libraries; (iv) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq and (v) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.
doi:10.1093/nar/gku818
PMCID: PMC4381050  PMID: 25223787
15.  Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain 
Neuron  2015;86(6):1369-1384.
SUMMARY
Neuronal diversity is essential for mammalian brain function but poses a challenge to molecular profiling. To address the need for tools that facilitate cell-type-specific epigenomic studies, we developed the first affinity purification approach to isolate nuclei from genetically defined cell types in a mammal. We combine this technique with next-generation sequencing to show that three subtypes of neocortical neurons have highly distinctive epigenomic landscapes. Over 200,000 regions differ in chromatin accessibility and DNA methylation signatures characteristic of gene regulatory regions. By footprinting and motif analyses, these regions are predicted to bind distinct cohorts of neuron subtype-specific transcription factors. Neuronal epigenomes reflect both past and present gene expression, with DNA hyper-methylation at developmentally critical genes appearing as a novel epigenomic signature in mature neurons. Taken together, our findings link the functional and transcriptional complexity of neurons to their underlying epigenomic diversity.
doi:10.1016/j.neuron.2015.05.018
PMCID: PMC4499463  PMID: 26087164
16.  Integrated Core Facility Support and Optimization of Next Generation Sequencing Technologies 
New DNA sequencing technologies present an exceptional opportunity for novel and creative applications with the potential for breakthrough discoveries. To support such research efforts, the Cornell University Life Sciences Core Laboratories Center has implemented the Illumina HiSeq 2000 and the Roche 454 GS FLX platforms as academic core facility shared research resources. We have established sample handling methods, LIMS tools and BioHPC informatics analysis pipelines in support of these new technologies. Our genomics core laboratory, in collaboration with our epigenomics core and bioinformatics core, provides sample preparation and data generation services and both project consultation and analysis support for a wide range of possible applications, including de novo or reference based genome assembly, detection of genetic variation, transcriptome sequencing, small RNA profiling, and genome-wide epigenomic measurements of methylation and protein-nucleic acid interactions. Implementation of next generation sequencing platforms as shared resources with multidisciplinary core facility support enables cost effective access and broad based use of these technologies.
PMCID: PMC3186497
17.  Using ChIP-Seq Technology to Generate High-Resolution Profiles of Histone Modifications 
The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within.
doi:10.1007/978-1-61779-316-5_20
PMCID: PMC4151291  PMID: 21913086
Chromatin immunoprecipitation; ChIP-seq; Next generation sequencing; Epigenomics; Histone modifications; IP-Star; ChIP robot
18.  Next-Generation Sequencing: Application in Liver Cancer—Past, Present and Future? 
Biology  2012;1(2):383-394.
Hepatocellular Carcinoma (HCC) is the third most deadly malignancy worldwide characterized by phenotypic and molecular heterogeneity. In the past two decades, advances in genomic analyses have formed a comprehensive understanding of different underlying pathobiological layers resulting in hepatocarcinogenesis. More recently, improvements of sophisticated next-generation sequencing (NGS) technologies have enabled complete and cost-efficient analyses of cancer genomes at a single nucleotide resolution and advanced into valuable tools in translational medicine. Although the use of NGS in human liver cancer is still in its infancy, great promise rests in the systematic integration of different molecular analyses obtained by these methodologies, i.e., genomics, transcriptomics and epigenomics. This strategy is likely to be helpful in identifying relevant and recurrent pathophysiological hallmarks thereby elucidating our limited understanding of liver cancer. Beside tumor heterogeneity, progress in translational oncology is challenged by the amount of biological information and considerable “noise” in the data obtained from different NGS platforms. Nevertheless, the following review aims to provide an overview of the current status of next-generation approaches in liver cancer, and outline the prospects of these technologies in diagnosis, patient classification, and prediction of outcome. Further, the potential of NGS to identify novel applications for concept clinical trials and to accelerate the development of new cancer therapies will be summarized.
doi:10.3390/biology1020383
PMCID: PMC3674503  PMID: 23750322
Hepatocellular carcinoma (HCC); Next-generation sequencing (NGS); personalized medicine; integrative genomics
19.  Next-Generation Sequencing: Application in Liver Cancer—Past, Present and Future? 
Biology  2012;1(2):383-394.
Hepatocellular Carcinoma (HCC) is the third most deadly malignancy worldwide characterized by phenotypic and molecular heterogeneity. In the past two decades, advances in genomic analyses have formed a comprehensive understanding of different underlying pathobiological layers resulting in hepatocarcinogenesis. More recently, improvements of sophisticated next-generation sequencing (NGS) technologies have enabled complete and cost-efficient analyses of cancer genomes at a single nucleotide resolution and advanced into valuable tools in translational medicine. Although the use of NGS in human liver cancer is still in its infancy, great promise rests in the systematic integration of different molecular analyses obtained by these methodologies, i.e., genomics, transcriptomics and epigenomics. This strategy is likely to be helpful in identifying relevant and recurrent pathophysiological hallmarks thereby elucidating our limited understanding of liver cancer. Beside tumor heterogeneity, progress in translational oncology is challenged by the amount of biological information and considerable “noise” in the data obtained from different NGS platforms. Nevertheless, the following review aims to provide an overview of the current status of next-generation approaches in liver cancer, and outline the prospects of these technologies in diagnosis, patient classification, and prediction of outcome. Further, the potential of NGS to identify novel applications for concept clinical trials and to accelerate the development of new cancer therapies will be summarized.
doi:10.3390/biology1020383
PMCID: PMC3674503  PMID: 23750322
Hepatocellular carcinoma (HCC); Next-generation sequencing (NGS); personalized medicine; integrative genomics
20.  Distinct Epigenomic Features in End-Stage Failing Human Hearts 
Circulation  2011;124(22):2411-2422.
Background
The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist.
Methods and Results
We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability.
Conclusions
Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy.
doi:10.1161/CIRCULATIONAHA.111.040071
PMCID: PMC3634158  PMID: 22025602
genes; genome-wide analysis; genomics; heart failure
21.  Multilayer-omics analyses of human cancers: exploration of biomarkers and drug targets based on the activities of the International Human Epigenome Consortium 
Epigenetic alterations consisting mainly of DNA methylation alterations and histone modification alterations are frequently observed in cancers associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, or with cigarette smoking. Accumulating evidence suggests that alterations of DNA methylation are involved even in the early and precancerous stages. On the other hand, in patients with cancers, aberrant DNA methylation is frequently associated with tumor aggressiveness and poor patient outcome. Recently, epigenome alterations have been attracting a great deal of attention from researchers who are focusing on not only cancers but also neuronal, immune and metabolic disorders. In order to accurately identify disease-specific epigenome profiles that could be potentially applicable for disease prevention, diagnosis and therapy, strict comparison with standard epigenome profiles of normal tissues is indispensable. However, epigenome mechanisms show heterogeneity among tissues and cell lineages. Therefore, it is not easy to obtain a comprehensive picture of standard epigenome profiles of normal tissues. In 2010, the International Human Epigenome Consortium (IHEC) was established to coordinate the production of reference maps of human epigenomes for key cellular states. In order to gain substantial coverage of the human epigenome, the IHEC has set an ambitious goal to decipher at least 1000 epigenomes within the next 7–10 years. We consider that pathway analysis using genes showing multilayer-omics abnormalities, including genome, epigenome, transcriptome, proteome and metabolome abnormalities, may be useful for elucidating the molecular background of pathogenesis and for exploring possible therapeutic targets for each disease.
doi:10.3389/fgene.2014.00024
PMCID: PMC3924033  PMID: 24592273
epigenetics; epigenome; DNA methylation; International Human Epigenome Consortium (IHEC); multilayer/integrated disease omics analyses
22.  Genetics, genomics and their relevance to pathology and therapy 
Genetic and genomic investigations are a starting point for the study of human disease, seeking to discover causative variants relevant to disease pathophysiology. Over the past 5 years, massively parallel, high-throughput, next-generation sequencing techniques have revolutionized genetics and genomics, identifying the causes of many mendelian diseases. The application of whole genome sequencing and whole exome sequencing to large populations has produced several publicly-available sequence datasets that have revealed the scope of human genetic variation, and have contributed to important methodological advances in the study of both common and rare genetic variants in genetically-complex diseases. The importance of noncoding genetic variation has been highlighted by the Encyclopedia of DNA Elements (ENCODE) project and NIH Roadmap Epigenomics Program, and integrated analyses of these datasets, together with disease-specific datasets, will provide an important and powerful tool for determining the mechanisms through which disease-associated, noncoding variation influence disease risk.
doi:10.1016/j.berh.2014.05.001
PMCID: PMC4149217  PMID: 24974057
pediatric rheumatology; somatic mosaicism; genomewide association study; targeted deep resequencing; miRNA; lncRNA
23.  Profiling genome-wide DNA methylation 
DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.
doi:10.1186/s13072-016-0075-3
PMCID: PMC4926291  PMID: 27358654
DNA methylation; Bisulfite sequencing; Hydroxymethylation; Single-cell; Methylome; WGBS; RRBS
24.  Emerging patterns of epigenomic variation 
Trends in genetics : TIG  2011;27(6):242-250.
Fuelled by new sequencing technologies, epigenome mapping projects are revealing epigenomic variation at all levels of biological complexity, from species to cells. Comparisons of methylation profiles among species reveal evolutionary conservation of gene body methylation patterns, pointing to the fundamental role of epigenomes in gene regulation. At the human population level, epigenomic changes provide footprints of the effects of genomic variants within the vast non-protein coding fraction of the genome while comparisons of the epigenomes of parents and their offspring point to quantitative epigenomic parent-of-origin effects confounding classical Mendelian genetics. At the organismal level, comparisons of epigenomes from diverse cell types provide insights into cellular differentiation. Finally, comparisons of epigenomes from monozygotic twins help dissect genetic and environmental influences on human phenotypes and longitudinal comparisons reveal aging-associated epigenomic drift. The development of new bioinformatic frameworks for comparative epigenome analysis is putting epigenome maps within reach of researchers across a wide spectrum of biological disciplines.
doi:10.1016/j.tig.2011.03.001
PMCID: PMC3104125  PMID: 21507501
25.  Genome-Wide Alteration of Histone H3K9 Acetylation Pattern in Mouse Offspring Prenatally Exposed to Arsenic 
PLoS ONE  2013;8(2):e53478.
Chronic exposure to arsenic in drinking water, especially in utero or perinatal exposure, can initiate neurological and cognitive dysfunction, as well as memory impairment. Several epidemiological studies have demonstrated cognitive and learning deficits in children with early exposure to low to moderate levels of arsenic, but pathogenic mechanisms or etiology for these deficits are poorly understood. Since in vivo studies show a role for histone acetylation in cognitive performance and memory formation, we examined if prenatal exposure to arsenic causes changes in the epigenomic landscape. We exposed C57Bl6/J mice to 100 μg/L arsenic in the drinking water starting 1 week before conception till birth and applied chromatin immunoprecipitation followed by high-throughput massive parallel sequencing (ChIP-seq) to evaluate H3K9 acetylation pattern in the offspring of exposed and control mice. Arsenic exposure during embryonic life caused global hypo-acetylation at H3K9 and changes in functional annotation with highly significant representation of Krüppel associated box (KRAB) transcription factors in brain samples from exposed pups. We also found that arsenic exposure of adult mice impaired spatial and episodic memory, as well as fear conditioning performance. This is the first study to demonstrate: a) genome wide changes in H3K9 acetylation pattern in an offspring prenatally exposed to arsenic, and b) a connection between moderate arsenic exposure and cognitive impairment in adult mice. The results also emphasize the applicability of Next Generation Sequencing methodology in studies aiming to reveal the role of environmental factors, other than dietary restriction, in developmental reprogramming through histone modifications during embryonic development.
doi:10.1371/journal.pone.0053478
PMCID: PMC3566160  PMID: 23405071

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