scholarly journals Experimental and Bioinformatic Approaches to Studying DNA Methylation in Cancer

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 349
Author(s):  
Angelika Merkel ◽  
Manel Esteller
Keyword(s):  
Dna Methylation ◽  
Cancer Research ◽  
Data Processing ◽  
Computational Analysis ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Primary Data ◽  
Cell Populations ◽  
Epigenetic Mark ◽  
Bioinformatic Approaches

DNA methylation is an essential epigenetic mark. Alterations of normal DNA methylation are a defining feature of cancer. Here, we review experimental and bioinformatic approaches to showcase the breadth and depth of information that this epigenetic mark provides for cancer research. First, we describe classical approaches for interrogating bulk DNA from cell populations as well as more recently developed approaches for single cells and multi-Omics. Second, we focus on the computational analysis from primary data processing to the identification of unique methylation signatures. Additionally, we discuss challenges such as sparse data and cellular heterogeneity.

scholarly journals scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

2021 ◽  
Author(s):  
Amanda Raine ◽  
Anders Lundmark ◽  
Alva Annett ◽  
Ann-Christin Wiman ◽  
Marco Cavalli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Single Cells ◽  
K562 Cells ◽  
Epigenetic Mark ◽  
Library Preparation ◽  
New Development ◽  
Liver Nuclei ◽  
Pooling Strategy ◽  
Methyl Cytosine

DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

scholarly journals Single-Cell DNA-Methylation Analysis Reveals Epigenetic Chimerism in Preimplantation Embryos

Science ◽  
2013 ◽  
Vol 341 (6150) ◽  
pp. 1110-1112 ◽  
Author(s):  
Chanchao Lorthongpanich ◽  
Lih Feng Cheow ◽  
Sathish Balu ◽  
Stephen R. Quake ◽  
Barbara B. Knowles ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Early Stage ◽  
Single Cells ◽  
Preimplantation Embryos ◽  
Epigenetic Mark ◽  
Epigenetic Alterations ◽  
Multiple Loci ◽  
Mammalian Embryos ◽  
Genomic Reprogramming ◽  
Dna Methylation Analysis

Epigenetic alterations are increasingly recognized as causes of human cancers and disease. These aberrations are likely to arise during genomic reprogramming in mammalian preimplantation embryos, when their epigenomes are most vulnerable. However, this process is only partially understood because of the experimental inaccessibility of early-stage embryos. Here, we introduce a methodologic advance, probing single cells for various DNA-methylation errors at multiple loci, to reveal failed maintenance of epigenetic mark results in chimeric mice, which display unpredictable phenotypes leading to developmental arrest. Yet we show that mouse pronuclear transfer can be used to ameliorate such reprogramming defects. This study not only details the epigenetic reprogramming dynamics in early mammalian embryos but also suggests diagnostic and potential future therapeutic applications.

scholarly journals Magnetic levitation of single cells

2015 ◽  
Vol 112 (28) ◽  
pp. E3661-E3668 ◽  
Author(s):  
Naside Gozde Durmus ◽  
H. Cumhur Tekin ◽  
Sinan Guven ◽  
Kaushik Sridhar ◽  
Ahu Arslan Yildiz ◽  
...  
Keyword(s):  
Magnetic Levitation ◽  
Single Cells ◽  
Nonsmall Cell Lung Cancer ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Label Free ◽  
Biological Characterization ◽  
Lung Cancer Cell ◽  
Cellular Density ◽  
Cellular Events

Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10−4g⋅mL−1. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

Novel Microtechnology System for Cytometric Analysis of Adherent Cell Populations

2010 ◽  
Author(s):  
Nicholas M. Gunn ◽  
Mark Bachman ◽  
Lifeng Zheng ◽  
G.-P. Li ◽  
Edward L. Nelson
Keyword(s):  
Single Cells ◽  
Pulsed Laser ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Microscope Slide ◽  
Glass Microscope Slide ◽  
Glass Microscope

The increasing appreciation of tissue cellular heterogeneity and recent identification of rare cell populations within tissues that are associated with specific biological behaviors, e.g., progenitor cells, has illuminated a limitation of current technologies to study such adherent cells directly from primary tissues. The micropallet array is a recently-developed technology designed to address this limitation by virtue of its capacity to isolate and recover single adherent cells on individual micropallets [1]. Micropallet arrays consist of hundreds of thousands of microscale polymer pedestals (“micropallets”) uniformly arrayed on a glass microscope slide. The micropallets are made from a high aspect photopolymerizable polymer using photolithographic methods. Cells are applied to the arrays and fall stochastically upon its surface, with single cells adhering to individual micropallets. Cells are then analyzed in situ and single, unperturbed cells can be selected and collected from the array by releasing the underlying micropallets using a focused pulsed laser.

Genome-scale sequence data processing and epigenetic analysis of DNA methylation

2013 ◽  
Vol 35 (6) ◽  
pp. 685-694
Author(s):  
Ting-Zhang WANG ◽  
Gao SHAN ◽  
Jian-Hong XU ◽  
Qing-Zhong XUE
Keyword(s):  
Dna Methylation ◽  
Data Processing ◽  
Sequence Data ◽  
Epigenetic Analysis ◽  
Genome Scale ◽  
Scale Sequence

scholarly journals Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sunny Z. Wu ◽  
Daniel L. Roden ◽  
Ghamdan Al-Eryani ◽  
Nenad Bartonicek ◽  
Kate Harvey ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Tumour Heterogeneity ◽  
Fresh Tissue ◽  
Human Cancers ◽  
Cryopreserved Cell ◽  
Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

scholarly journals Cellular lensing and near infrared fluorescent nanosensor arrays to enable chemical efflux cytometry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Soo-Yeon Cho ◽  
Xun Gong ◽  
Volodymyr B. Koman ◽  
Matthias Kuehne ◽  
Sun Jin Moon ◽  
...  
Keyword(s):  
Near Infrared ◽  
Single Cells ◽  
Microfluidic Channel ◽  
Human Monocyte ◽  
Cellular Heterogeneity ◽  
Label Free ◽  
Nanotube Array ◽  
Chemical Cytometry ◽  
Rich Information ◽  
Non Destructive

AbstractNanosensors have proven to be powerful tools to monitor single cells, achieving spatiotemporal precision even at molecular level. However, there has not been way of extending this approach to statistically relevant numbers of living cells. Herein, we design and fabricate nanosensor array in microfluidics that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). nIR fluorescent carbon nanotube array is integrated along microfluidic channel through which flowing cells is guided. We can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR profiles and extract rich information. This unique biophotonic waveguide allows for quantified cross-correlation of biomolecular information with various physical properties and creates label-free chemical cytometer for cellular heterogeneity measurement. As an example, the NCC can profile the immune heterogeneities of human monocyte populations at attomolar sensitivity in completely non-destructive and real-time manner with rate of ~600 cells/hr, highest range demonstrated to date for state-of-the-art chemical cytometry.

scholarly journals Cellular Heterogeneity–Adjusted cLonal Methylation (CHALM) improves prediction of gene expression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jianfeng Xu ◽  
Jiejun Shi ◽  
Xiaodong Cui ◽  
Ya Cui ◽  
Jingyi Jessica Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cellular Heterogeneity ◽  
Biological Functions ◽  
Global Correlation ◽  
Differentially Methylated Genes ◽  
Promoter Dna Methylation ◽  
Functional Consequences ◽  
Promoter Dna ◽  
Underlying Mechanisms

AbstractPromoter DNA methylation is a well-established mechanism of transcription repression, though its global correlation with gene expression is weak. This weak correlation can be attributed to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity–Adjusted cLonal Methylation (CHALM) as a methylation quantification method. CHALM improves understanding of the functional consequences of DNA methylation, including its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method enables detection of differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms.

scholarly journals RA3 is a reference-guided approach for epigenetic characterization of single cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shengquan Chen ◽  
Guanao Yan ◽  
Wenyu Zhang ◽  
Jinzhao Li ◽  
Rui Jiang ◽  
...  
Keyword(s):  
Single Cell ◽  
Computational Analysis ◽  
Reference Data ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Biological Variation ◽  
Superior Performance ◽  
High Dimensionality ◽  
High Degree

AbstractThe recent advancements in single-cell technologies, including single-cell chromatin accessibility sequencing (scCAS), have enabled profiling the epigenetic landscapes for thousands of individual cells. However, the characteristics of scCAS data, including high dimensionality, high degree of sparsity and high technical variation, make the computational analysis challenging. Reference-guided approaches, which utilize the information in existing datasets, may facilitate the analysis of scCAS data. Here, we present RA3 (Reference-guided Approach for the Analysis of single-cell chromatin Accessibility data), which utilizes the information in massive existing bulk chromatin accessibility and annotated scCAS data. RA3 simultaneously models (1) the shared biological variation among scCAS data and the reference data, and (2) the unique biological variation in scCAS data that identifies distinct subpopulations. We show that RA3 achieves superior performance when used on several scCAS datasets, and on references constructed using various approaches. Altogether, these analyses demonstrate the wide applicability of RA3 in analyzing scCAS data.

UPAH MENGUPAH PERTANIAN DALAM TINJAUAN FIQH MUAMALAH (Studi di Nagari Bukit Kandung Kecamatan X Koto Diatas Kabupaten Solok)

2021 ◽  
Vol 2 (3) ◽  
pp. 59
Author(s):  
Susanti Krismon ◽  
Syukri Iska
Keyword(s):  
Data Collection ◽  
Data Processing ◽  
Field Research ◽  
Secondary Data ◽  
Research Field ◽  
Farm Workers ◽  
Data Sources ◽  
Primary Data

This article discusses the implementation of wages in agriculture in Nagari Bukit Kandung Subdistrict X Koto Atas, Solok Regency in a review of muamalah fiqh. The type of research is field research (field research). The data sources consist of primary data sources, namely from farmers and farm laborers who were carried out to 8 people and 4 farm workers, while the secondary data were obtained from documents in the form of the Bukit Kandung Nagari Profile that were related to this research, which could provide information or data. Addition to strengthen the primary data. Data collection techniques that the author uses are observation, interviews and documentation. The data processing that the author uses is qualitative. Based on the results of this study, the implementation of wages in agriculture carried out in Nagari Bukit Kandung District X Koto Diatas Solok Regency is farm laborers who ask for their wages to be given in advance before they carry out their work without an agreement to give their wages at the beginning. Because farm laborers ask for their wages to be given at the beginning, many farm workers work not as expected by farmers and there are also farm workers who are not on time to do the work that should be done. According to the muamalah fiqh review, the implementation of wages in agriculture in Nagari Bukit Kandung is not allowed because there is an element of gharar in the contract and there are parties who are disadvantaged in the contract, namely the owner of the fields.

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