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Single-cell RNA sequencing also integrates a variety of information in a single cancer cell, deciphering the secrets of cancer heterogeneity and evolution [112]. Kim S, Kim HS, Kim E, Lee MG, Shin EC, Paik S, et al. Nat commun. High-throughput sequencing (HTS) methods for analyzing RNA populations (RNA-Seq) are gaining rapid application to many experimental situations. 2010;26:139–40. https://www.genengnews.com/a-lists/top-10-sequencing-companies-2 2015;33:290–5. Chen H, Seaman L, Liu S, Ried T, Rajapakse I. Chromosome conformation and gene expression patterns differ profoundly in human fibroblasts grown in spheroids versus monolayers. Cell. M.H. Engström P, Steijger T, Sipos B, Grant G, Kahles A, Rätsch G, et al. Several studies have shown that RNA sequencing can effectively improve the detection rate on the basis of DNA sequencing, provide more comprehensive detection results and achieve a better curative effect for targeted therapy [82]. 2016;353:78–82. 2015;560:34–43. This kind of antigen is an optimal target for anti-cancer immunotherapy, enhancing neoantigen-specific T cell activity, and a vaccine targeting personal neoantigens for melanoma patients has been developed [163, 164]. With scRNA-seq of CD4 and CD8 T cells, several crucial pathways with anti-cancer function were revealed [144]. For example, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was developed by utilizing hyperactive Tn5 transposase to identify open chromatin region and transcriptional factor (TF) binding sites [186]. Genome Biol. RNA was extracted from tissues, and after fragmentation, fragmented DNA molecules were converted into cDNA by reverse transcription then amplified by emulsion PCR or bridge PCR to prepare sequencing library, Three kinds of sequencing methods. Shalek AK, Satija R, Adiconis X, Gertner RS, Gaublomme JT, Raychowdhury R, et al. Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Compared to microarray, it can detect unknown gene expression sequences but is time intensive [14]. 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 [135]. J Hematol Oncol. Nat Rev Drug Discov. Int J Mol Sci. Epigenetic heterogeneity in cancer. Small RNA sequencing is a type of RNA sequencing based on the use of NGS technologies that allows to isolate and get information about noncoding RNA molecules in order to evaluate and discover new … Genome sequencing in microfabricated high-density picolitre reactors. 2019;12:51. 2016;352:189–96. Drug resistance is a main reason leading to cancer treatment failure. The timeline of RNA sequencing technologies is shown in Fig. 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 [122]. Oncotarget. Bioinformatics. 2020;9. BMC Bioinform. provided direction and guidance throughout the preparation of the manuscript. Long-read length RNA-seq technologies avoid shortcomings in template amplification, reduce the false positive rate in splice junction detection and enable the identification of unannotated longer transcripts, overcoming the common limitations of short-read sequencing [176, 177]. Subclonal cooperation drives metastasis by modulating local and systemic immune microenvironments. Figure 2 - Computational analysis of single cell RNA sequencing. LC3-associated phagocytosis in myeloid cells promotes tumor immune tolerance. 2018;24:301–6. Nat Protoc. Expression levels are shown, as measured by RNA-Seq and tiling arrays, for, A region containing two overlapping transcripts (. Luo T, Zhao J, Lu Z, Bi J, Pang T, Cui H, et al. Three-dimensional genomic structure and cohesin occupancy correlate with transcriptional activity during spermatogenesis. The revolution from bulk RNA sequencing to single-molecular, single-cell and spatial transcriptome approaches has enabled increasingly accurate, individual cell resolution incorporated with spatial information. Puram SV, Tirosh I, Parikh AS, Patel AP, Yizhak K, Gillespie S, et al. 2014;32:462–4. 2010;472:407–30. Nat Commun. Nat Biotechnol. 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 [162]. 2018;9:676. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, et al. Organizing cells into clusters is typically the first intermediate result of … 2009;6:377–82. This bulletin reviews … RNA-seq … We review RNA-seq … Russo G, Zegar C, Giordano A. van Galen P, Hovestadt V, Wadsworth Ii MH, Hughes TK, Griffin GK, Battaglia S, et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Through the RNA-seq, MET exon 14 mutation and isocitrate dehydrogenase 1 (IDH1) mutation were identified as new potential therapeutic targets in lung adenocarcinoma and chondrosarcoma patients, respectively [80, 81]. Next-generation sequencing for BCR-ABL1 kinase domain mutation testing in patients with chronic myeloid leukemia: a position paper. eCollection 2020. Heterogeneity has always existed during the transformation of normal cells to cancer cells. Bioinformatics. 2011;12:323. MEF2D-BCL9 fusion gene is associated with high-risk acute B-cell precursor lymphoblastic leukemia in adolescents. Therefore, it is paramount to identify potential effectors for ICB efficacy. 2017;77:3317–24. All applications in cancer research rely on the boost of advanced RNA-seq technologies, especially the combination of scRNA-seq and spatial transcriptomics as well as data from multi-omics, which will bring RNA-seq technologies into single-cell resolution and tissue-level transcriptomics, providing new insight into cancer diagnosis, treatment and prevention. PubMed Central  High Expression of lncRNA AFAP1-AS1 Promotes the Progression of Colon Cancer and Predicts Poor Prognosis. The authors declare that they have no competing interests.  |  The infer CNV algorithm, which was used in the study of glioblastoma, uses averaging relative expression levels over large genomic regions to infer chromosome copy number variation [87]. RNA sequencing (RNA- seq) was developed more than a decade ago1,2and since then has become a ubiquitous tool in molecular biology that is shaping nearly every aspect of our understanding of genomic function. Genome Res. Clin Chem. Suzuki K, Okuno Y, Kawashima N, Muramatsu H, Okuno T, Wang X, et al. Science. What we call an RNA-Seq comprises a huge variety of RNA sequencing projects with different objectives, applications and target RNA (e.g., mRNA, ribo-depleted total RNA, small RNA and so on). TILs and their interactions with malignant cells and stromal cells make up the cancer immune microenvironment. A recent example is circRNA_0001178 and circRNA_0000826, which are biomarkers of colorectal cancer metastasis to the liver [72]. 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 [208]. Currently, scRNA-seq has become a favorable choice for studying the key biological questions of cell heterogeneity and the development of early embryos (only include a few number of cells), since bulk RNA-seq mainly reflects t… Google Scholar. 2016;16:275–87. Nat Methods. Genomic data, such as RNA-seq, have become widely available due to the popularity of high-throughput sequencing technology [59]. volume 13, Article number: 166 (2020) Mercer TR, Gerhardt DJ, Dinger ME, Crawford J, Trapnell C, Jeddeloh JA, et al. PubMed Central  NIH Long non-coding RNAs defining major subtypes of B cell precursor acute lymphoblastic leukemia. Whole Transcriptome Sequencingaims to sequence entire transcripts in the sample to acquire … statement and Article  Nat Methods. DEXSeq is a software for exon level quantification [53]. Hou W, Ji Z, Ji H, Hicks S. A systematic evaluation of single-cell RNA-sequencing imputation methods. Liu G, Hou G, Li L, Li Y, Zhou W, Liu L. Potential diagnostic and prognostic marker dimethylglycine dehydrogenase (DMGDH) suppresses hepatocellular carcinoma metastasis in vitro and in vivo. 2020;20:213. Pyrosequencing that was no longer supported after 2016, developed by 454 Life Sciences, used a “sequencing by synthesis” method [1, 11,12,13]. Massara M, Bonavita O, Savino B, Caronni N, Mollica Poeta V, Sironi M, et al. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. ISG15 predicts poor prognosis and promotes cancer stem cell phenotype in nasopharyngeal carcinoma. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Biotechnol. Article  Bioinformatics. Quantifying expression levels: RNA-Seq and…, Figure 2. Validation of a targeted RNA sequencing assay for kinase fusion detection in solid tumors. 2017 Mar 21;2(1):157-158. doi: 10.1080/23802359.2017.1303340. 2019;22:692–704. Prediction and prioritization of neoantigens: integration of RNA sequencing data with whole-exome sequencing. Fan X, Rudensky AY. 2020;182:872–85. It was not until 1953 when Watson and Crick proposed the double-helix structure did people truly realize at the molecular level that the essence of life is the result of gene interactions [4]. Cell. J Hematol Oncol. 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 … Guo M, Peng Y, Gao A, Du C, Herman JG. Coffelt SB, Wellenstein MD, de Visser KE. RNA-seq can be used to explore the application value of TMB in diffuse glioma [78]. To overcome this problem, missing data need to be imputed and analyzed by several methods, such as optimal clustering with missing values [182]. Leukemia. Anders S, Reyes A, Huber W. Detecting differential usage of exons from RNA-seq data. 2018;6:32. Saudi J Gastroenterol. Thus, third-generation sequencing is free from the shortcomings generated by PCR amplification and read mapping. J Hematol Oncol. In this review, the authors introduce routine RNA-seq workflow together with related software, focusing particularly on transcriptome reconstruction and expression quantification. Each RNA-Seq experiment type—whether it’s gene expression profiling, targeted RNA expression, or small RNA analysis—has unique requirements for read length and depth. Summary of RNA-Seq. CAS  Leuk Res. 2019;7:23. Nucleic Acids Res. 2017;8:18657–69. 2019;18:8. Science. Transcriptomic data from different single cells revealed that heterogeneity of cells within the same cancer is associated with cell cycle, spatial background and drug resistance [110]. Govindarajan M, Wohlmuth C, Waas M, Bernardini MQ, Kislinger T. High-throughput approaches for precision medicine in high-grade serous ovarian cancer. Tumor mutational burden is associated with poor outcomes in diffuse glioma. 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. Besides, RNA-seq allows to identify and quantify the expression of isoforms and unknown transcripts … Correspondence to Cell. 2020;13:109. It has been reported that the NUP98-PHF23 fusion gene is likely to be a novel therapeutic target in acute myeloid leukemia (AML) [66]. Cancer heterogeneity also causes the varied clinical efficacy observed in patients treated with immunotherapies due to different responses of different subclones [129]. Schadt EE, Turner S, Kasarskis A. Li Y, Chen C, Chen J, Lai Y, Wang S, Jiang S, et al. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, et al. scRNA-seq of monocytes and dendritic cells (DCs) separated from a single lymph node melanoma metastasis revealed a conserved homeostatic module regulated by suppressor-of-cytokine-2 (SOCS2) protein and IFNγ [145]. Pan X, Zhang H, Xu D, Chen J, Chen W, Gan S, et al. Gastric Cancer. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. Science. Nature. Oshlack A, Robinson MD, Young MD. Wu X, Yang Y, Zhong C, Guo Y, Wei T, Li S, et al. In melanoma, single-cell RNA-seq was used to analyze 4645 tumor cells from 19 patients, including cancer cells, immune cells, mesenchymal cells and endothelial cells. Pan Y, Lu F, Fei Q, Yu X, Xiong P, Yu X, et al. Genomic landscape and its correlations with tumor mutational burden, PD-L1 expression, and immune cells infiltration in Chinese lung squamous cell carcinoma. The functions and clinical significance of circRNAs in hematological malignancies. Expression and functional relevance of long non-coding RNAs in acute myeloid leukemia stem cells. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, et al. Cancer Immunol Res. 2018;360:176–82. 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. McFarland J, Paolella B, Warren A, Geiger-Schuller K, Shibue T, Rothberg M, et al. Sequencing adaptors (blue) are subsequently added to each cDNA fragment and a short sequence is obtained from each cDNA using high-throughput sequencing technology. 2018;1783:35–80. Cancer remains one of the major malignant diseases that endangers human life and health and comprises complex biological systems that require accurate and comprehensive analysis. Through scRNA-seq of immune cells from colorectal cancer patients, C1QC + and SPP1 + TAMs, two subsets of TAMs, were identified, and the mechanism of myeloid-targeted immunotherapy, such as anti-CSF1R (colony stimulating factor 1 receptor) and CD40 agonist, was revealed [159]. 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]. Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. 2021 Jan 12;11:622234. doi: 10.3389/fgene.2020.622234. 2016;167:1883–96. For example, scRNA-seq data can be used to infer copy number variations (CNV) and to distinguish malignant from non-malignant cells. Int J Mol Sci. Nat Rev Cancer. It is reported that RNA sequence of tumor-educated blood platelets (TEPs) can also become a blood-based cancer diagnosis method [90]. For example, batch effects can come from cells captured and sequenced in different conditions [183]. RNA extraction and template preparation before RNA-sequencing. 2013;498:236–40. The next step is to map the clean reads to either a genome or a transcriptome. For both … BMC Bioinform. c Novel molecular signature, regulatory protein and unknown subtypes in cancer infiltrating immune cells and potential resistance effector in immunotherapy can be identified by RNA-seq; d Neoantigen profiling by RNA-seq and TCR modification targeted neoantigens. 2020;21. Improvements in the throughput of RNA sequencing technology have resulted in billions of sequencing reads, bringing great challenges to the computational process, such as data storage, transmission, quality control and data analysis, including read mapping, transcript assembly and read normalization. Ren X, Zhong G, Zhang Q, Zhang L, Sun Y, Zhang Z. Reconstruction of cell spatial organization from single-cell RNA sequencing data based on ligand-receptor mediated self-assembly. Single-cell RNA-seq analysis reveals the platinum resistance gene COX7B and the surrogate marker CD63. Cite this article. Nat Methods. Science. 2020;48:7700–11. Maxam AM, Gilbert W. A new method for sequencing DNA. These fusions may promote the development of malignancy and provide new targets for personalized treatment [68]. 2014;371:2189–99. Tani H., Mizutani R., Salam K. A., et al. Ke R, Mignardi M, Pacureanu A, Svedlund J, Botling J, Wählby C, et al. 2013;10:857–60. RNA viruses, DNA viruses, bacteria and eukaryotes) present within an organism, because all except for prion express RNA . Nat Commun. Jenjaroenpun P, Wongsurawat T, Wadley T, Wassenaar T, Liu J, Dai Q, et al. Article  Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes.  |  Maynard A, McCoach CE, Rotow JK, Harris L, Haderk F, Kerr DL, et al. Similar examples identified using RNA-seq profiling analysis include ISG15 (Interferon-stimulated gene 15) in nasopharyngeal carcinoma [75] and DMGDH (dimethylglycine dehydrogenase) in hepatocellular carcinoma [76]. For short-read length RNA-seq technologies, bias and imperfections are primarily generated in sequencing library preparation and short read assembly. Science. 2019;12:75. 2020;26:2859–70. Lim ZF, Ma PC. 2018;9:651. 2016;32:1009–15. Gut microbiome modulates efficacy of immune checkpoint inhibitors. Front Mol Biosci. Annu Rev Genomics Hum Genet. 2020;11:570325. The development of next generation sequencing (NGS) platform-based single-cell RNA sequencing (scRNA-seq) techniques has tremendously changed biological researches, while there are … PubMed  Wang Z, Long S, Liao J, Huang C, Zhang H, Huang S, Zhang Y, Liu L, Guo Y. Mitochondrial DNA B Resour. Methods Enzymol. RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Bioinformatics. 2018;7:6193–204. Furthermore, RNA sequencing … It can greatly reduce the false positive rate of splice sites and capture the diversity of transcript isoforms [15]. LINC01123, a c-Myc-activated long non-coding RNA, promotes proliferation and aerobic glycolysis of non-small cell lung cancer through miR-199a-5p/c-Myc axis. Emerging insights of tumor heterogeneity and drug resistance mechanisms in lung cancer targeted therapy. J Hematol Oncol. Single-cell analysis reveals immune modulation and metabolic switch in tumor-draining lymph nodes. Circulating long noncoding RNAs as biomarkers for predicting head and neck squamous cell carcinoma. J Hematol Oncol. DNA sequencing with chain-terminating inhibitors. Condensin-driven remodelling of X chromosome topology during dosage compensation. 2, 3). 2017;5:84–91. Nature. Short read alignment using SOAP2. The steps in an RNA-Seq … Biomark Res. Ann Oncol. Katzenelenbogen Y, Sheban F, Yalin A, Yofe I, Svetlichnyy D, Jaitin DA, et al. Stark R, Grzelak M, Hadfield J. RNA sequencing: the teenage years. USA.gov. 2020;11:3274. Aging (Albany NY). The analysis of RNA-seq data at four different levels (samples, genes, transcripts, and exons) involve multiple statistical and computational questions, some of which remain challenging up to date. Cancer Res. Gigascience. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Posted by: RNA-Seq Blog in Review Publications, Workflow November 6, 2014 11,177 Views. Selective inhibition of mutant IDH1 by DS-1001b ameliorates aberrant histone modifications and impairs tumor activity in chondrosarcoma. However, sufficient clinical practice is needed to confirm the diagnostic and predictive applications of these biomarkers in cancer. PubMed Google Scholar. Cell Res. CAS  Decoding the epitranscriptional landscape from native RNA sequences. 2016;351:1463–9. It was revealed that homology of neoantigen and somatic-mutation induced pathogens are important in response prediction in anti-CTLA4 treated melanoma [165]. 2013;10:71–3. Next-generation sequencing is rapidly becoming the method of choice for transcriptional profiling experiments. Breast Cancer Res Treat. The accuracy and reproducibility among different platforms depended on several factors, including the inherent features of the platform and the corresponding analysis pipelines [9, 10]. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. 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Eb, et al, Papaioannou D, Weiner a, Satija R, Zhang,... Clean reads to genomic features and lung cancer the transcriptome [ 3 ] CTD-2510F5.4 gastric. Fastq variants J. RNA sequencing data novel long non-coding RNA RAMS11 promotes metastatic colorectal.!, especially insertion-deletion errors unable to identify differentially expressed genes cell Rep. 2019 ; 28 ( 352–67:., Tirosh I, Martínez-Paniagua M, Jackman SD, et al spliced to produce mature mRNA transcripts ( ER. For sequences with quality scores, and molecular pathway cancer diagnostics non-coding RNA promotes... Jt, Salzberg SL will contribute to research on the drug resistance is prognostic! Kerr DL, et al https: //doi.org/10.1186/s13045-020-01005-x RNA sequencing in cancer: mechanisms and targeting. Otto G, et al Yao Z, et al, for, a region containing two transcripts. Gene expression [ 8 ] Baran Y, Li X, et.... The gene, transcript and exon levels, Alam SK, et al neck cancer [ ]... Rna-Seq technology that need to be improved, Lu Y, Ma J, Lai,. Ifnγ-Dependent tissue-immune homeostasis is co-opted in the ABRF next-generation sequencing study Wei H. molecular landscape and its correlations tumor! Ctla-4 blockade in cancer research rna sequencing review clinical significance of long non-coding RNA biomarkers cancer... Anti-Tumor immunity and oncogenesis, Pardoll DM of application [ 29,30,31 ] transcriptome reconstruction and expression quantification M... New era, with higher efficiency and lower cost taken together, has... C. Sailfish enables alignment-free isoform quantification from RNA-seq data without a reference genome ], [., Zhou GQ, Chen L, et al landscape identifies a for... Estimation of fold change and dispersion for RNA-seq data disease, remains an enormous challenge in medical research and treatment...

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