In this review, the latest studies on RNA sequencing technology and their applications in cancer are summarized, and future challenges and opportunities for RNA sequencing technology in cancer applications are discussed. Through RNA-seq, it has been found that non-coding RNAs and fusion genes play an important role in mediating the drug resistance of hematological malignancies . Chen J, Suo S, Tam PP, Han JJ, Peng G, Jing N. Spatial transcriptomic analysis of cryosectioned tissue samples with geo-seq. Heterogeneity has always existed during the transformation of normal cells to cancer cells. 2020;11:570325. Wang Z, Qin B. Prognostic and clinicopathological significance of long noncoding RNA CTD-2510F5.4 in gastric cancer. Cancer Immunol Res. 2018;174:1293–308. Seo JS, Ju YS, Lee WC, Shin JY, Lee JK, Bleazard T, et al. Another immunotherapy, myeloid-targeted immunotherapy, is based on the complexity of tumor-infiltrating myeloid cells, including DCs and tumor-associated macrophages (TAMs) revealed by scRNA-seq . 2013;29:15–21. Subclonal cooperation drives metastasis by modulating local and systemic immune microenvironments. Defining a personal, allele-specific, and single-molecule long-read transcriptome. The Basic Framework of Single-Cell RNA-Sequencing Methods To sequence mRNA from a single cell, one has to overcome two challenges that are not present in standard population-level methods: (1) capturing single cells and (2) amplification of minute amounts of mRNA from a cell… Moon M, Nakai K. Stable feature selection based on the ensemble L (1)-norm support vector machine for biomarker discovery. The advent of single-cell RNA sequencing (scRNA-seq) provides unprecedented opportunities for exploring gene expression profile at the single-cell level. J Hematol Oncol. Nat Commun. 2021 Jan 27;21(1):64. doi: 10.1186/s12870-020-02761-1. Many studies have shown that gene fusions are closely related to oncogenesis and are appreciated as both ideal cancer biomarkers and therapeutic targets . Longitudinal single-cell RNA sequencing of patient-derived primary cells reveals drug-induced infidelity in stem cell hierarchy. 2013;14:R36. Clin Cancer Res. RNA sequencing became a vital tool for revealing the mechanisms of cancer drug resistance. Sharma A, Cao EY, Kumar V, Zhang X, Leong HS, Wong AML, et al. PubMed A recent single-cell RNA-seq study of 49 samples of metastatic lung cancer revealed changes in plasticity induced by non-small cell lung cancer treatment, providing new directions for clinical treatment . Higher intratumor neoantigen heterogeneity and clonal neoantigen burden increases sensitivity to ICB and contributes to better clinical outcome in patients with melanoma and advanced non-small cell lung cancer . Cell Rep. 2019;27:708–18. Role of microRNAs, circRNAs and long noncoding RNAs in acute myeloid leukemia. 2020;9:1830513. 2015;33:290–5. Compared with scRNA-seq, another emerging technology spatial transcriptome sequencing incorporates information on the spatial location of cells. b The heterogeneity and drug resistance of cancer cells identified by RNA-seq. Science. Maynard A, McCoach CE, Rotow JK, Harris L, Haderk F, Kerr DL, et al. Furthermore, various new technologies based on RNA-seq have been developed for specific applications. 2020;16:3149–62. As an essential part of high-throughput sequencing, RNA sequencing (RNA-seq), especially single-cell RNA sequencing (scRNA-seq), provides biological information on a single tumor cell, analyzes the determinants of intratumor expression heterogeneity and identifies the molecular basis of formation of many oncological diseases [2, 3… eCollection 2020. Manage cookies/Do not sell my data we use in the preference centre. Cancer infiltrating T cells also play an anti-tumor role through impairment of an autophagy protein, LC3 (microtubule-associated protein 1A/1B-light chain 3, often short for LC3)-associated phagocytosis (LAP), demonstrating the role of autophagy in oncogenesis and suppression revealed by scRNA-seq . Topalian SL, Taube JM, Anders RA, Pardoll DM. Bioinformatics. 2018;7:1–6. Nat Biotechnol. PubMed Central Simonis M, Kooren J, de Laat W. An evaluation of 3C-based methods to capture DNA interactions. 2019;79:1362–77. BMC Bioinform. Ke R, Mignardi M, Pacureanu A, Svedlund J, Botling J, Wählby C, et al. 2014;371:2189–99. 1977;74:560–4. eCollection 2020. From RNA-seq reads to differential expression results. 4. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq. Mingye Hong and Shuang Tao have contributed equally to this work, Institute of Laboratory Medicine, Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, School of Medical Technology, Guangdong Medical University, Dongguan, 523808, China, Mingye Hong, Xuanmei Huang, Shaohui Huang & Hua Zhang, Biotherapy Center, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510630, China, Shuang Tao, Li-Ting Diao, Shu-Juan Xie & Zhen-Dong Xiao, Health Science Center, The University of Texas, Houston, 77030, USA, You can also search for this author in PubMed Cell. The emerging technology of RNA sequencing will contribute to research on cancer heterogeneity and evolution. Bioinformatics. In the cancer immune microenvironment, neutrophils, in addition to T cells, are also key components of cancer progression and cancer drug resistance [138,139,140,141]. Ponzetta A, Carriero R, Carnevale S, Barbagallo M, Molgora M, Perucchini C, et al. J Hematol Oncol. Sequencing by Oligonucleotide Ligation and Detection, Pacific Biosciences single-molecule real-time, Single-cell combinatorial indexing RNA sequencing, Split-pool ligation-based transcriptome sequencing, Human epidermal growth factor receptor type 2, Brother of the regulator of imprinted sites, Microtubule-associated protein 1A/1B-light chain 3-associated phagocytosis, Cytotoxic T-lymphocyte-associated protein 4, Assay for Transposase-Accessible Chromatin using sequencing, Carbon copy chromosome conformation capture, Clustered regularly interspaced short palindromic repeats. Single-cell RNA sequencing was first published in 2009 to profile the transcriptome at single-cell resolution . Google Scholar. Azizi E, Carr AJ, Plitas G, Cornish AE, Konopacki C, Prabhakaran S, et al. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 2010;28:511–5. PD-1 blockade in tumors with mismatch-repair deficiency. 2019;12:42. Gene. It has been reported that the NUP98-PHF23 fusion gene is likely to be a novel therapeutic target in acute myeloid leukemia (AML) . Science. J Hematol Oncol. This collection contains protocol diagrams, advantages and disadvantages, and related peer-reviewed publications … Another technology, snRNA-seq, is invaluable for detecting cellular heterogeneity of cancer and has been employed to identification of a sub-population of adipocytes regulating cancer genesis . A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. 2020;9. Pan Y, Lu F, Fei Q, Yu X, Xiong P, Yu X, et al. Yang H, Sun L, Guan A, Yin H, Liu M, Mao X, et al. J Mol Diagn. Pyrosequencing that was no longer supported after 2016, developed by 454 Life Sciences, used a “sequencing by synthesis” method [1, 11,12,13]. Yu J, Jiang PYZ, Sun H, Zhang X, Jiang Z, Li Y, et al. 2010;19:R227–40. Nature. And their different mechanisms are shown in detail. USA.gov. Wculek SK, Malanchi I. Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Google Scholar. Li S, Tighe S, Nicolet C, Grove D, Levy S, Farmerie W, et al. 2020;13:134. Hicks S, Townes F, Teng M, Irizarry R. Missing data and technical variability in single-cell RNA-sequencing experiments. 2015;8:69. 2018;19:93–109. RNA sequencing (RNA-seq) provides a quantitative and open system for profiling transcriptional outcomes on a large scale and therefore facilitates a large diversity of applications, including basic … Li Z, Song W, Rubinstein M, Liu D. Recent updates in cancer immunotherapy: a comprehensive review and perspective of the 2018 China Cancer Immunotherapy Workshop in Beijing. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Google Scholar. L.Z., L-T. D., X-M. H., S-H. H., S-J. HHS Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Notably, bias could be produced due to differences in read data handling, necessitating the improvement of current bioinformatics pipelines. Zhu L, Li J, Gong Y, Wu Q, Tan S, Sun D, et al. Unruh D, Zewde M, Buss A, Drumm MR, Tran AN, Scholtens DM, et al. Jaitin D, Weiner A, Yofe I, Lara-Astiaso D, Keren-Shaul H, David E, et al. Cunha LD, Yang M, Carter R, Guy C, Harris L, Crawford JC, et al. J Hematol Oncol. J Hematol Oncol. Boluki S, Zamani Dadaneh S, Qian X, Dougherty E. Optimal clustering with missing values. A map of tumor-host interactions in glioma at single-cell resolution. BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. A survey of best practices for RNA-seq data analysis. Biomark Res. Sci Rep. 2020;10:1535. By applying both RNA sequencing and small RNA sequencing, a study on pancreatic cancer identified differential expression of simple repetitive sequences (SSRs) and demonstrated that the frequency of SSR motifs changed dramatically, which is expected to become a tumor biomarker . Drop-Seq and InDrop were initially reported in 2015 by analyzing mouse retina cell and embryonic stem cell transcriptomes, identifying novel cell types. In recent years, single-nucleus RNA sequencing (snRNA-seq) has also received extensive attention due to its solving the problem that single-cell RNA sequencing cannot be applied to frozen specimens and cannot obtain all cell types in a given tissue [114, 115]. 2020;13:36. 2019;7:23. Cell. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Proc Natl Acad Sci USA. The immune microenvironment and neoantigen landscape of aggressive salivary gland carcinomas differ by subtype. 2016;7:32607–16. 2014;32:462–4. Nucleic Acids Res. Nat Commun. The first step of data analysis is to assess and clean the raw sequencing data, which is usually provided in the form of FASTQ files . 2019;85:106198. In genome sequencing, fragmented genomic DNA are sequenced and whole genome is assembled from the reads sequence. Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, et al. Transl Oncol. A systematic analysis of oncogenic gene fusions in primary colon cancer. 2020;182(1232–51):e22. Organizing cells into clusters is typically the first intermediate result of … Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Davies KD, Lomboy A, Lawrence CA, Yourshaw M, Bocsi GT, Camidge DR, et al. A novel androgen-reduced prostate-specific lncRNA, PSLNR, inhibits prostate-cancer progression in part by regulating the p53-dependent pathway. It can greatly reduce the false positive rate of splice sites and capture the diversity of transcript isoforms . 2020;181:442–59. 2020;11:4296. 2016;353:78–82. Cancer Cell. 2018;11:1080–9. It should be noted that the lack of detailed functional implications of the identified RNAs in platelets in the field of platelet RNA research is also an urgent problem to be solved . Advances in targeted therapy for acute myeloid leukemia. Circulating long noncoding RNAs as biomarkers for predicting head and neck squamous cell carcinoma. Single-molecule sequencing: sequence methods to enable accurate quantitation. Identification of a novel cancer stem cell subpopulation that promotes progression of human fatal renal cell carcinoma by single-cell RNA-seq analysis. This highly conserved homeostatic program establishes a connection between autoimmune prevention and immune surveillance in cancer . An immunogenic personal neoantigen vaccine for patients with melanoma. Nat Commun. Through scRNA-seq of murine sarcomas and certain human cancers, neutrophils with CSF3R (colony stimulating factor 3 receptor) expression were found to be a part of type 1 antitumor immunity associated with unconventional CD4−CD8−αβ T cells (UTCαβ) in anti-cancer immunity, indicating better prognosis . Or a transcriptome from RNA-seq reads using lightweight algorithms analyzing mouse retina cell and embryonic stem cell subpopulation promotes! Venteicher as, Hebert C, Xu R, Yang X, Jiang J, et al Sahu,! 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