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      Complex Analysis of Single-Cell RNA Sequencing Data

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          Abstract

          Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell–cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

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          Most cited references149

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          Is Open Access

          Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

          In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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            The Sequence Alignment/Map format and SAMtools

            Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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              Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

              Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
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                Author and article information

                Contributors
                khozyainova@onco.tnimc.ru
                Journal
                Biochemistry (Mosc)
                Biochemistry (Mosc)
                Biochemistry. Biokhimiia
                Pleiades Publishing (Moscow )
                0006-2979
                1608-3040
                10 March 2023
                2023
                : 88
                : 2
                : 231-252
                Affiliations
                [1 ]GRID grid.473330.0, ISNI 0000 0004 5932 2274, Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, ; 634050 Tomsk, Russia
                [2 ]GRID grid.14476.30, ISNI 0000 0001 2342 9668, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, ; 119991 Moscow, Russia
                [3 ]GRID grid.14476.30, ISNI 0000 0001 2342 9668, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, ; 119991 Moscow, Russia
                [4 ]Laboratory of Artificial Intelligence and Bioinformatics, The Russian Clinical Research Center for Gerontology, Pirogov Russian National Medical University, 129226 Moscow, Russia
                [5 ]GRID grid.14476.30, ISNI 0000 0001 2342 9668, School of Public Administration, Lomonosov Moscow State University, ; 119991 Moscow, Russia
                [6 ]GRID grid.14476.30, ISNI 0000 0001 2342 9668, Faculty of Fundamental Medicine, Lomonosov Moscow State University, ; 119991 Moscow, Russia
                [7 ]Research Institute of Personalized Medicine, National Center for Personalized Medicine of Endocrine Diseases, National Medical Research Center for Endocrinology, 117036 Moscow, Russia
                [8 ]GRID grid.77602.34, ISNI 0000 0001 1088 3909, Laboratory of Complex Analysis of Big Bioimage Data, National Research Tomsk State University, ; 634050 Tomsk, Russia
                [9 ]GRID grid.465331.6, Department of Oncohematology, Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, ; 117198 Moscow, Russia
                [10 ]GRID grid.4886.2, ISNI 0000 0001 2192 9124, Laboratory of Bioinformatics and Molecular Genetics, Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, ; 119334 Moscow, Russia
                Article
                2453
                10.1134/S0006297923020074
                10000364
                37072324
                1bfb0d57-0db1-48cd-8b72-9a5c2dbaa4a8
                © Pleiades Publishing, Ltd. 2023

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 23 September 2022
                : 13 December 2022
                : 13 December 2022
                Categories
                Review
                Custom metadata
                © Pleiades Publishing, Inc. 2023

                single-cell rna sequencing,cell cycle,clustering,differential expression,cell type,trajectory inference,cell–cell interaction,gene regulatory network,copy number variation,single nucleotide variant,phylogenetics,epigenomics,spatial transcriptomics

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