single cell Bisulfite-seq

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization...

Data from both bulk and single-cell whole-genome DNA methylation experiments are under-utilized in many ways. This is attributable to inefficient mapping of methylation sequencing reads, routinely discarded genetic information, and neglected read-level epigenetic and genetic linkage information. We introduce the BISulfite-seq Command line User Interface Toolkit (BISCUIT) and its companion R/Bioconductor package, biscuiteer, for simultaneous extraction of genetic and epigenetic information from bulk and single-cell DNA methylation sequencing...

Epigenetic mechanisms, such as DNA methylation, are crucial factors in animal development. In some mammals, almost all DNA methylation is erased during embryo development and re-established in a sex- and cell-specific manner. This erasure and re-establishment is thought to primarily be a vertebrate-specific trait. Insects are particularly interesting in terms of development as many species often undergo remarkable morphological changes en route to maturity, that is, morphogenesis. However, little is known about the role of epigenetic mechanisms in this process across species...

Penile squamous cell carcinoma (SCC) is a rare and aggressive tumour mainly related to lifestyle behaviour and human papillomavirus (HPV) infection. Environmentally induced loss of imprinting (LOI) at the H19 differentially methylated region (H19DMR) is associated with many cancers in the early events of tumorigenesis and may be involved in the pathogenesis of penile SCC. We sought to evaluate the DNA methylation pattern at H19DMR and its association with HPV infection in men with penile SCC by bisulfite sequencing (bis-seq)...

BACKGROUND: Detecting human cancers through cell-free DNA (cfDNA) in blood is a sensitive and non-invasive option. However, capturing multiple forms of epigenetic information remains a technical and financial challenge. METHODS: To address this, we developed multimodal epigenetic sequencing analysis (MESA), a flexible and sensitive approach to capturing and integrating a diverse range of epigenetic features in cfDNA using a single experimental assay, i.e., non-disruptive bisulfite-free methylation sequencing, such as Enzymatic Methyl-seq...

MOTIVATION: The utilization of single-cell bisulfite sequencing (scBS-seq) methods allows for precise analysis of DNA methylation patterns at the individual cell level, enabling the identification of rare populations, revealing cell-specific epigenetic changes, and improving differential methylation analysis. Nonetheless, the presence of sparse data and an overabundance of zeros and ones, attributed to limited sequencing depth and coverage, frequently results in reduced precision accuracy during the process of differential methylation detection using scBS-seq...

Pseudouridine (Ψ) is an abundant RNA modification that is present in and affects the functions of diverse non-coding RNA species, including rRNA, tRNA and small nuclear RNA. Ψ also exists in mammalian mRNA and probably exhibits functional roles; however, functional investigations of mRNA Ψ modifications in mammals have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed bisulfite-induced deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution...

Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A toward 5mC and chemically protected 5hmC...

Epstein-Barr virus (EBV) is associated with 10% of human gastric carcinomas, which are distinguished by a CpG island methylator phenotype. In gastric carcinoma tumors and cell lines, the EBV genome also exhibits a high degree of 5-methyl cytosine (5mC) marks, which are propagated by host DNA methyltransferases (DNMT) with each cell cycle. Therefore, we sought to determine the effect of DNMT inhibition by the small molecule Decitabine (DCB) on EBV genomic 5mC and chromatin structure in two tumor-derived gastric cancer cell lines, YCCEL1 and SNU719...

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury. To investigate the role of DNA methylation in spinal cord injury, we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points (day 0-42) after spinal cord injury in mice. Global DNA methylation levels, specifically non-CpG (CHG and CHH) methylation levels, decreased modestly following spinal cord injury...

INTRODUCTION: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. RESULTS: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line...

Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration. To characterize Casuarina genomic diversity, we sequenced and constructed de novo genome assemblies of the three most widely planted species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome-scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi-C)...

BACKGROUND: Advanced maternal age (AMA) has increased in many high-income countries in recent decades. AMA is generally associated with a higher risk of various pregnancy complications, and the underlying molecular mechanisms are largely unknown. In the current study, we profiled the DNA methylome of 24 human chorionic villi samples (CVSs) from early pregnancies in AMA and young maternal age (YMA), 11 CVSs from early spontaneous abortion (SA) cases using reduced representation bisulfite sequencing (RRBS), and the transcriptome of 10 CVSs from AMA and YMA pregnancies with mRNA sequencing(mRNA-seq)...

Single-cell Nucleosome Occupancy and Methylome sequencing (scNOMe-seq) is a multimodal assay that simultaneously measures endogenous DNA methylation and nucleosome occupancy (i.e., chromatin accessibility) in single cells. scNOMe-seq combines the activity of a GpC Methyltransferase, an enzyme which methylates cytosines in GpC dinucleotides, with bisulfite conversion, whereby unmethylated cytosines are converted into thymines. Because GpC Methyltransferase acts only on cytosines present in non-nucleosomal regions of the genome, the subsequent bisulfite conversion step not only detects the endogenous DNA methylation, but also reveals the genome-wide pattern of chromatin accessibility...

OBJECTIVE: Haematogenous dissemination is a prevalent route of colorectal cancer (CRC) metastasis. However, as the gatekeeper of vessels, the role of tumour pericytes (TPCs) in haematogenous metastasis remains largely unknown. Here, we aimed to investigate the heterogeneity of TPCs and their effects on CRC metastasis. DESIGN: TPCs were isolated from patients with CRC with or without liver metastases and analysed by single-cell RNA sequencing (scRNA-seq). Clinical CRC specimens were collected to analyse the association between the molecular profiling of TPCs and CRC metastasis...

Multiomics data integration is one of the leading frontiers of complex disease research and integrative biology. The advances in single-cell sequencing technologies offer yet another crucial dimension in multiomics research. The single-cell studies enable the study and integration of multiomics data simultaneously in the same cell. We report in this study multiomics data integration in single-cell resolution using Bayesian networks (BNs) in a case study of hepatocellular carcinoma (HCC). A BN encodes the conditional dependencies/independencies of variables using a graphical model with an accompanying joint probability...

BACKGROUND: High-throughput sequencing of blood cell-free DNA (cfDNA) techniques offer an opportunity to characterize and monitor cancer rapidly in a non-invasive and real-time manner. Nonetheless, there lacks a tool within therapeutic arsenal to identify multi-omics alterations simultaneously from a single biopsy. In current times, bisulfite-based sequencing detects 5mC and 5hmC at single-base resolution is the golden standard of DNA methylation, while the degradation of DNA and biased sequencing data are the problems of this method...

BACKGROUND: Epstein-Barr virus (EBV) latent infection is associated with genome-wide epigenomic changes in several malignancies, but its role in epigenetic dysregulation remains unclear in nasopharyngeal carcinoma (NPC). METHODS: To investigate EBV-associated epigenetic dysregulation, we performed a multi-omics study by integrating whole-genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin using sequencing (ATAC-Seq), whole-exome sequencing (WES), and single-cell RNA sequencing (scRNA-Seq) data...

Single-cell DNA methylation profiling currently suffers from excessive noise and/or limited cellular throughput. We developed scTAM-seq, a targeted bisulfite-free method for profiling up to 650 CpGs in up to 10,000 cells per experiment, with a dropout rate as low as 7%. We demonstrate that scTAM-seq can resolve DNA methylation dynamics across B-cell differentiation in blood and bone marrow, identifying intermediate differentiation states that were previously masked. scTAM-seq additionally queries surface-protein expression, thus enabling integration of single-cell DNA methylation information with cell atlas data...

DNA methylation is one of the most studied epigenetic modifications that has applications ranging from transcriptional regulation to aging, and can be assessed by bisulfite sequencing (BS-seq) or enzymatic methyl sequencing (EM-seq) at single base-pair resolution. The permutations of methylation statuses given by aligned reads reflect the methylation patterns of individual cells. These patterns at specific genomic locations are sought to be indicative of cellular heterogeneity within a cellular population, which are predictive of developments and diseases; therefore, methylation heterogeneity has potentials in early detection of these changes...