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Climate change, hydro-power dams and socio-economic development in the upper Mekong River, have caused changes in surface water resources and agricultural transformation in the Vietnamese Mekong Delta (VMD). It is unpredictable and influences decision-making processes on water resource management. This study aims at applying the DAPP (Dynamic Adaptive Policy Pathways) approach to develop solution roadmaps supporting rice production in the Mekong Delta projection to 2030 under the context of uncertain changes on surface water resources. Data were collected from local officers, farmers, experts at Can Tho University, Technical University Delft, and Utrecht University using semi-structured interview techniques including workshops, group discussions, and in-depth interviews. The consolidated solutions from stakeholder’s opinions were evaluated at different socio-economic and environmental dimensions, specially focused on the time of tipping point of each solution. The study results show that two solution roadmaps were developed as sample scenarios to promote rice production in the upper area of the VMD. The determination of the solutions roadmaps based on the agreement of the stakeholders, which is a new feature of this approach in linking many stakeholders for the general development of the region. The DAPP approach could be suitable to support decision-making on surface water resources management in the context of uncertain changes. Therefore, further studies can apply this approach to other regions in the VMD which will support to reinforce and demonstrate the applicability of the DAPP approach on supporting decision-making in the face of increasingly uncertain changes in the VMD.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations.Implications:TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS.Visual Overview:http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

A) Expression of hemoglobin genes which increase in terminally differentiated erythroid cells. B) Expression of key erythrocyte marker genes. TPM=Transcripts per kilobase million; a gene expression measure for RNAseq that normalizes for gene-length and the sample sequencing depth.

A) Dose-response curve of wild-type TF1 cell viability for three different lengths of 5-Aza treatments (1 dose per day for a total length of treatment between 1 and 3 days). Points represent replicates per condition, shaded region corresponds to 95% confidence band estimated by 4 parameter logistic regression model (EC50 and slope as parameter and lower bounded at zero; modeled with R package "drc"). B) Dose-response of DNMT1 downregulation 24 hours after the indicated 5-Aza dose. C) Time course of DNMT1 downregulation following the indicated dosage schedules.

Cells were treated with 100uM L-AA or vehicle 24hrs before DNA isolation. A) 5hmC dot blot for L-AA treated or untreated Cells. B) Standard curve of 5hmC densitometric signal vs. pmol (calculated from 5hmC standard with known 5hmC amount). C) Quantification of dot blot signal for TET2-WT and KO cells +/- L-AA.

Line plots in grid: each colored line represents a single CpG locus over time after 5-Aza treatment. Black line indicates mean value for that cluster group. Dotted black line indicates mean +/- 1 standard deviation. From left to right: clustering solutions with K=2 to K=9 clusters (derived from cutting the hierarchical cluster tree at different levels); top to bottom: the patterns of methylation in each cluster for that cluster solution. Mean silhouette width plot shows the mean silhouette width of all clusters for each individual clustering solution. K=4 clusters produced the most well-defined clusters as indicated by the peak in silhouette width.

Comprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment.

Here we describe a computational protocol for performing quality control analysis on shallow sequencing data obtained from 10X snRNA-seq experiments. The workflow starts with raw MiSeq run folders and uses cellranger to generate count matrices. The raw count matrices are analyzed and sequencing saturation plots are generated. The saturation plots are then compared against plots from a reference set of libraries with varying qualities (bad, fair, good, great), thus allowing for the determination of sequencing requirements as well as an assessment of the overall quality of each 10X snRNA experiment.

Eyewitness misidentification accounts for 70% of verified erroneous convictions. To address this alarming phenomenon, research has focused on factors that influence likelihood of correct identification, such as the manner in which a lineup is conducted. Traditional lineups rely on overt eyewitness responses that confound two covert factors: strength of recognition memory and the criterion for deciding what memory strength is sufficient for identification. Here we describe a lineup that permits estimation of memory strength independent of decision criterion. Our procedure employs powerful techniques developed in studies of perception and memory: perceptual scaling and signal detection analysis. Using these tools, we scale memory strengths elicited by lineup faces, and quantify performance of a binary classifier tasked with distinguishing perpetrator from innocent suspect. This approach reveals structure of memory inaccessible using traditional lineups and renders accurate identifications uninfluenced by decision bias. The approach furthermore yields a quantitative index of individual eyewitness performance., Eyewitness errors contribute to wrongful convictions. Here, the authors present a lineup procedure that reveals the structure of eyewitness memory, reduces decision bias, and measures performance of individual witnesses.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. 10x Genomics Single Cell ATAC sequencing is a microdroplet-based method that allows for the effective capture, sequencing and profiling of accessible chromatin in single nuclei. Chromatin accessibility is a major determinant of gene regulation, defining the transcriptional regulatory networks that determine cellular identity and function as well as additional biological processes (e.g., differentiation, proliferation, development and responses to the extracellular environment). The 10X Multiome ATAC + Gene Expression assay permits capture of both RNA expression and epigenomic profiles from the same nuclei for a deeper understanding of cell type or state gene regulation. Here we present a modified version of the published 10X protocol [5] that we have adapted for the processing of nuclei isolated from adult human organs or tissues (e.g. kidney). Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. This 10X Multiome ATAC + Gene Expression protocol permits the generation of a molecular atlas of a human organ or tissue with comprehensive cell types and minimal processing artifacts. References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2. 5. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev D).

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., An examination of motor cortex in humans, marmosets and mice reveals a generally conserved cellular makeup that is likely to extend to many mammalian species, but also differences in gene expression, DNA methylation and chromatin state that lead to species-dependent specializations.

Understanding kidney disease relies upon defining the complexity of cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods. We have applied multiple single-cell or -nucleus assays (>400,000 nuclei/cells) and spatial imaging technologies to a broad spectrum of healthy reference (n = 42) and disease (n = 42) kidneys. This has provided a high resolution cellular atlas of 100 cell types that include rare and novel cell populations. The multi-omic approach provides detailed transcriptomic profiles, epigenomic regulatory factors, and spatial localizations for major cell types spanning the entire kidney. We further identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments. Molecular signatures of these states permitted their localization within injury neighborhoods using spatial transcriptomics, and large-scale 3D imaging analysis of ∼1.2 million neighborhoods provided linkages to active immune responses. These analyses further defined biological pathways relevant to injury niches, including signatures underlying the transition from reference to predicted maladaptive states that were associated with a decline in kidney function during chronic kidney disease. This human kidney cell atlas, including injury cell states and neighborhoods, will be a valuable resource for future studies.

10x Genomics Single Cell ATAC (v1) sequencing is a microdroplet-based method that allows for the effective capture, sequencing and profiling of accessible chromatin in single nuclei. Chromatin accessibility is a major determinant of gene regulation, defining the transcriptional regulatory networks that determine cellular identity and function as well as additional biological processes (e.g., differentiation, proliferation, development and responses to the extracellular environment). Chromatin accessibility sequencing (ATAC-seq) provides insight into the upstream regulatory landscape associated with open and accessible chromatin regions. References 1. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 2. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 3. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2. 4. Chromium Single Cell ATAC Reagent Kits User Guide v1 (Rev B) CG000168, support.10xgenomics.com. 5. Lake and Zhang. Nuclei Isolation Protocol Applicable to Adult Human Kidney Tissues, kpmp.org/researcher-resources.

Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6051.

BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 699.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations. Implications: TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Comprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment.

Ninh Kieu District is located in the center of Can Tho City, established under the Government’s Decree No. 05/2004/ND-CP. This is a modern and rich district, with a history of formation and development associated with the Mekong Delta. According to statistics in 2019, Ninh Kieu District has an average population of 280,792 people, with a population density of 9,605 people/km2 and an area of 29.23 km2. In the current period, the urbanization process in Ninh Kieu District takes place at a fast and strong pace. This has made it impossible for the urban drainage system to keep up with this change, thereby creating great difficulties for the drainage management here. In order to deal with these enormous challenges, the managers in this area need to incorporate new technologies into drainage management. This paper presents the main result of building a spatial database to support drainage management for Ninh Kieu District. This is considered as a fundamental source of data for application of GIS in urban drainage research and management in Ninh Kieu District.

10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. Here we present a modified version of the published 10X protocol [1] that we have adapted for the processing of adult human kidney nuclei. References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2.

Recurrent mutations implicate several epigenetic regulators in the early molecular pathobiology of myelodysplastic syndromes (MDS). We hypothesized that MDS subtypes defined by DNA methylation (DNAm) patterns could enhance our understanding of MDS disease biology and identify patients with convergent epigenetic profiles. Bisulfite padlock probe sequencing was used to measure DNAm of ∼500 000 unique cytosine guanine dinucleotides covering 140 749 nonoverlapping regulatory regions across the genome in bone marrow DNA samples from 141 patients with MDS. Application of a nonnegative matrix factorization (NMF)–based decomposition of DNAm profiles identified 5 consensus clusters described by 5 NMF components as the most stable grouping solution. Each of the 5 NMF components identified by this approach correlated with specific genetic abnormalities and categorized patients into 5 distinct methylation clusters, each largely defined by a single NMF component. Methylation clusters displayed unique differentially methylated regulatory loci enriched for active and bivalent promoters and enhancers. Two clusters were enriched for samples with complex karyotypes, although only one had an increased number of TP53 mutations. Each of the 3 most frequently mutated splicing factors, SF3B1, U2AF1, and SRSF2, was enriched in different clusters. Mutations of ASXL1, EZH2, and RUNX1 were coenriched in the SRSF2-containing cluster. In multivariate analysis, methylation cluster membership remained independently associated with overall survival. Targeted DNAm profiles identify clinically relevant subtypes of MDS not otherwise distinguished by mutations or clinical features. Patients with diverse genetic lesions can converge on common DNAm states with shared pathogenic mechanisms and clinical outcomes.

Bisulfite padlock probes (BSPP) are a method for the targeted quantification of DNA methylation in mammalian genomes. They can simultaneously characterize the level of methylcytosine modification in a large number of targeted regions at single-base resolution. A major advantage of BSPP is that it allows the flexible capture of an arbitrary subset of genomic regions (hundreds to hundreds of thousands of genomic loci) in single-tube reactions. Large number of samples can be processed efficiently and converted into multiplexed sequencing libraries with only three enzymatic steps, without the conventional library preparation procedures. BSPP are applicable to clinical studies, screening cell lines, and for quantifying low abundance regions using deep sequencing.

Bisulfite padlock probes (BSPP) are a method for the targeted quantification of DNA methylation in mammalian genomes. They can simultaneously characterize the level of methylcytosine modification in a large number of targeted regions at single-base resolution. A major advantage of BSPP is that it allows the flexible capture of an arbitrary subset of genomic regions (hundreds to hundreds of thousands of genomic loci) in single-tube reactions. Large number of samples can be processed efficiently and converted into multiplexed sequencing libraries with only three enzymatic steps, without the conventional library preparation procedures. BSPP are applicable to clinical studies, screening cell lines, and for quantifying low abundance regions using deep sequencing.

Chemical modifications of DNA have been recognized as key epigenetic mechanisms for maintenance of the cellular state and memory. Such DNA modifications include canonical 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Recent advances in detection and quantification of DNA modifications have enabled epigenetic variation to be connected to phenotypic consequences on an unprecedented scale. These methods may use chemical or enzymatic DNA treatment, may be targeted or non-targeted and may utilize array-based hybridization or sequencing. Key considerations in the choice of assay are cost, minimum sample input requirements, accuracy and throughput. This Review discusses the principles behind recently developed techniques, compares their respective strengths and limitations and provides general guidelines for selecting appropriate methods for specific experimental contexts.

Adjacent CpG sites in mammalian genomes can be co-methylated due to the processivity of methyltransferases or demethylases. Yet discordant methylation patterns have also been observed, and found related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that exhibit highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks (MHBs) with 61 sets of whole genome bisulfite sequencing (WGBS) data, and further validated with 101 sets of reduced representation bisulfite sequencing (RRBS) data and 637 sets of methylation array data. Using a metric called methylation haplotype load (MHL), we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 cancer patients using methylation haplotypes.

A loss-of-function approach in mice is used to show that the methylcytosine dioxygenase Tet1 has a role in regulating meiosis and meiotic gene activation in female germ cells; Tet1 deficiency does not greatly affect genome-wide demethylation but has a more specific effect on the expression of a subset of meiotic genes. DNA methylation on cytosine is an important epigenetic modification, and the mechanisms controlling 5-methylcytosine (5mC) dynamics constitute an active area of research. The Tet family of dioxygenases can catalyse oxidation of 5mC to produce derivatives such as 5-hydroxymethylcytosine (5hmC), but little is known about the biological function of Tet proteins. Here, a loss-of-function approach in mice is used to show that Tet1 has a role in meiosis and meiotic gene activation in female germ cells. Tet1 deficiency does not greatly impact genome-wide demethylation, but has a more specific effect on the expression of a subset of meiotic genes. Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction1. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps2,3, including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA4,5. Although several epigenetic regulators, such as Dnmt3l and the histone methyltransferases G9a and Prdm9, have been reported to be crucial for meiosis6, little is known about how the expression of meiotic genes is regulated and how their expression contributes to normal meiosis. Using a loss-of-function approach in mice, here we show that the 5mC-specific dioxygenase Tet1 has an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation that takes place in primordial germ cells, but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus establishes a function for Tet1 in meiosis and meiotic gene activation in female germ cells.

Summary Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal, in colon, lung, breast, thyroid, and Wilms tumors, with intermediate variation in adenomas. Whole genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. These data suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer and could contribute to tumor heterogeneity.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.