DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2–5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.
Introduction: Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Because of the overwhelming use of SARS-CoV-2 reverse transcription (RT)-PCR tests worldwide, the availability of test kits has become a major bottleneck and the need to increase testing throughput is rising. We aim to overcome these challenges by pooling samples together, and performing RNA extraction and RT-PCR in pools. Methods: We tested the efficiency and sensitivity of pooling strategies for RNA extraction and RT-PCR detection of SARS-CoV-2. We tested 184 samples both individually and in pools to estimate the effects of pooling. We further implemented Dorfman pooling with a pool size of eight samples in large-scale clinical tests. Results: We demonstrated pooling strategies that increase testing throughput while maintaining high sensitivity. A comparison of 184 samples tested individually and in pools of eight samples showed that test results were not significantly affected. Implementing the eight-sample Dorfman pooling to test 26 576 samples from asymptomatic individuals, we identified 31 (0.12%) SARS-CoV-2 positive samples, achieving a 7.3-fold increase in throughput. Discussion: Pooling approaches for SARS-CoV-2 testing allow a drastic increase in throughput while maintaining clinical sensitivity. We report the successful large-scale pooled screening of asymptomatic
Pooling multiple swab samples prior to RNA extraction and real-time reverse-transcription (RT-PCR) analysis has been proposed as a strategy to reduce costs and increase throughput of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution, the rate of false positives, the actual efficiency (number of tests saved by pooling), and the impact of infection rate in the population on assay performance. Here we report an analysis of 133,816 samples collected between April-September 2020 and tested by Dorfman pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the frequently changing prevalence (0.5%-6%). We observed pooling efficiency and sensitivity that exceeded theoretical predictions, which resulted from the non-random distribution of positive samples in pools. Overall, our findings support the use of pooling for efficient large-scale SARS-CoV-2 testing.
MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the expression and translation of genes in healthy and diseased tissues. Herein, we characterize short RNAs from human HeLa cells found in the supraspliceosome, a nuclear dynamic machine in which pre-mRNA processing occurs. We sequenced small RNAs (<200 nt) extracted from the supraspliceosome, and identified sequences that are derived from 200 miRNAs genes. About three quarters of them are mature miRNAs, whereas the rest account for various defined regions of the pre-miRNA, and its hairpin-loop precursor. Out of these aligned sequences, 53 were undetected in cellular extract, and the abundance of additional 48 strongly differed from that in cellular extract. Notably, we describe seven abundant miRNA-derived sequences that overlap non-coding exons of their host gene. The rich collection of sequences identical to pre-miRNAs at the supraspliceosome suggests overlooked nuclear functions. Specifically, the abundant hsa-mir-99b may affect splicing of LINC01129 primary transcript through base-pairing with its exon-intron junction. Using suppression and overexpression experiments, we show that hsa-mir-7704 negatively regulates the level of the lncRNA HAGLR. We claim that in cases of extended base-pairing complementarity, such supraspliceosomal pre-miRNA sequences might have a role in transcription attenuation, maturation and processing.
MicroRNAs (miRNAs) act as negative regulators of gene expression in the cytoplasm. Previous studies have identified the presence of miRNAs in the nucleus. Here we study human breast cancer-derived cell-lines (MCF-7 and MDA-MB-231) and a non-tumorigenic cell-line (MCF-10A) and compare their miRNA sequences at the spliceosome fraction (SF). We report that the levels of miRNAs found in the spliceosome, their identity, and pre-miRNA segmental composition are cell-line specific. One such miRNA is miR-7704 whose genomic position overlaps HAGLR, a cancer-related lncRNA. We detected an inverse expression of miR-7704 and HAGLR in the tested cell lines. Specifically, inhibition of miR-7704 caused an increase in HAGLR expression. Furthermore, elevated levels of miR-7704 slightly altered the cell-cycle in MDA-MB-231. Altogether, we show that SF-miR-7704 acts as a tumor-suppressor gene with HAGLR being its nuclear target. The relative levels of miRNAs found in the spliceosome fractions (e.g., miR-100, miR-30a, and let-7 family) in non-tumorigenic relative to cancer-derived cell-lines was monitored. We found that the expression trend of the abundant miRNAs in SF was different from that reported in the literature and from the observation of large cohorts of breast cancer patients, suggesting that many SF-miRNAs act on targets that are different from the cytoplasmic ones. Altogether, we report on the potential of SF-miRNAs as an unexplored route for cancerous cell state.
Pooling multiple swab samples prior to RNA extraction and RT-PCR analysis was proposed as a strategy to reduce costs and increase throughput of SARS-CoV-2 tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution; the rate of false positives; the actual efficiency (number of tests saved by pooling) and the impact of infection rate in the population on assay performance. Here we report analysis of 133,816 samples collected at April-September 2020, tested by pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the reality of frequently changing prevalence rate (0.5%-6%). Surprisingly, we observed pooling efficiency and sensitivity that exceed theoretical predictions, which resulted from non-random distribution of positive samples in pools. Overall, the findings strongly support the use of pooling for efficient large high throughput SARS-CoV-2 testing.
Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.
DNA methylation is a fundamental epigenetic mark that governs chromatin organization, cell identity, and gene expression. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 207 healthy tissue samples. Replicates of the same cell-type are >99.5% identical, demonstrating robustness of cell identity programs to genetic variation and environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny, and identifies methylation patterns retained since gastrulation. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hyper-methylated loci are rare and are enriched for CpG islands, polycomb targets, and CTCF binding sites, suggesting a novel role in shaping cell type-specific chromatin looping. The atlas provides an essential resource for interpretation of disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.