Cited by 2,135Genomics and Phylogenetic StudiesSingle-cell and spatial transcriptomicsRNA Research and Splicing

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RNA sequencing: advances, challenges and opportunities

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Ozsolak, Fatih, Milos, Patrice M.·2011·Nature Reviews Genetics

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Abstract

In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.

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Influential
Effects of a wide range of dietary forage-to-concentrate ratios on nutrient utilization and hepatic transcriptional profiles in limit-fed Holstein heifers
Shi, H., Zhang, J., Li, S., Ji, S., Cao, Z., Zhang, H., & Wang, Y. (2018) · BMC Genomics · doi:10.1186/s12864-018-4529-9 · Cited by 31
“High-throughput RNA sequencing (RNA-Seq) is rapidly emerging as a favored approach for transcriptomeoriented studies, and can be used to analyze changes in gene expression across the entire transcriptome [15, 16].”
Elevated TOP2A and UBE2C expressions correlate with poor prognosis in patients with surgically resected lung adenocarcinoma: a study based on immunohistochemical analysis and bioinformatics
Guo, W., Sun, S., Guo, L., Song, P., Xue, X., Zhang, H., Zhang, G., Wang, Z., Qiu, B., Tan, F., et al. (2020) · Journal of Cancer Research and Clinical Oncology · doi:10.1007/s00432-020-03147-4 · Cited by 32
Tanshinone IIA Regulates Keap1/Nrf2 Signal Pathway by Activating Sestrin2 to Restrain Pulmonary Fibrosis
Li, H., Wu, M., Guo, C., Zhai, R., & Chen, J. (2022) · The American Journal of Chinese Medicine · doi:10.1142/s0192415x22500914 · Cited by 34
Nutritional systems biology of type 2 diabetes
Zhao, Y., Barrere‐Cain, R., & Yang, X. (2015) · Genes & Nutrition · doi:10.1007/s12263-015-0481-3 · Cited by 31
“More recently, sequencing-based RNA-Seq has become a revolutionary approach to transcriptome proﬁling because it is more sensitive and has a broader dynamic range than microarray tools (Ozsolak and Milos 2011; Wang et al. 2009).”
Longissimus lumborum muscle transcriptome analysis of Laiwu and Yorkshire pigs differing in intramuscular fat content
Chen, W., Fang, G.-F., Wang, S.-D., et al. (2017) · Genes & Genomics · doi:10.1007/s13258-017-0540-9 · Cited by 31
Acceleration of Nucleotide Semi-Global Alignment with Adaptive Banded Dynamic ProgrammingPreprint
Suzuki, H., & Kasahara, M. (2017) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/130633 · Cited by 32
“…start with aligning reads against themselves (for de novo assembly in whole genome re-sequencing research; Chin et al. (2013), Koren et al. (2017)) or reference sequences (for other reference-guided analyses, e.g. exome sequencing and RNA-seq; Pabinger et al. (2014), Ozsolak and Milos (2011)).”
Background
Three-dimensional assessments are necessary to determine the true, spatially resolved composition of tissues
Forjaz, A., Vaz, E., Matos-Romero, V., Joshi, S., Queiroga, V., Braxton, A. M., Jiang, A. C., Fujikura, K., Cornish, T. C., Hong, S., et al. (2025) · Cell Reports Methods · doi:10.1016/j.crmeth.2025.101075 · Cited by 31
Advances in bacterial transcriptome and transposon insertion-site profiling using second-generation sequencing
Febrer, M., McLay, K., Cáccamo, M., Twomey, K. B., & Ryan, R. P. (2011) · Trends in biotechnology · doi:10.1016/j.tibtech.2011.06.004 · Cited by 30
Comparative genomics approaches to understanding and manipulating plant metabolism
Bradbury, L. M., Niehaus, T. D., & Hanson, A. D. (2012) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2012.07.005 · Cited by 30
Diminishing returns in next-generation sequencing (NGS) transcriptome data
Lei, R., Ye, K., Gu, Z., & Sun, X. (2014) · Gene · doi:10.1016/j.gene.2014.12.013 · Cited by 30
“Regulatory diversity in the transcriptome, such as regulatory mutations or perturbed signaling pathways, often leads to phenotypic or functional differences including diseases (Sul et al., 2009; Marguerat and Bahler, 2010; Ozsolak and Milos, 2011).”
Background
Application of microarray analysis of foodborne Salmonella in poultry production: A review
Ricke, S. C., Khatiwara, A., & Kwon, Y. M. (2013) · Poultry Science · doi:10.3382/ps.2012-02740 · Cited by 30
“ther advancement of high-throughput sequencing technologies has led to methods that can generate the sequence of the entire transcriptome (RNA sequencing or RNA-Seq), thus enabling discovery of novel transcripts and uncovering sequence variations (Wang et al., 2009; Ozsolak and Milos, 2011; Raz et al., 2011; Mutz et a…”
Background
From -omics to personalized medicine in nephrology: integration is the key
Pesce, F., Pathan, S., & Schena, F. P. (2012) · Nephrology Dialysis Transplantation · doi:10.1093/ndt/gfs483 · Cited by 30
“Transcriptomics has been mainly based on microarrays so far, while measuring RNA by NGS is an unbiased method for genome-wide transcription factor binding-site profiling and transcription characterization that is called RNA-Seq [18].”
Method
De NovoAssembly of the Pea (Pisum sativumL.) Nodule Transcriptome
Zhukov, V. A., Zhernakov, A. I., Алексеевна, К. О., et al. (2015) · International Journal of Genomics · doi:10.1155/2015/695947 · Cited by 30
“As an alternative to whole genome sequencing, analysis of transcriptomes by RNA sequencing can provide extensive information about expressed genes [11, 12].”
Background
Identification of a putative polyketide synthase gene involved in usnic acid biosynthesis in the lichen Nephromopsis pallescens
Wang, Y., Geng, C., Yuan, X.-L., et al. (2018) · PLoS ONE · doi:10.1371/journal.pone.0199110 · Cited by 30
A New Machine Learning-Based Framework for Mapping Uncertainty Analysis in RNA-Seq Read Alignment and Gene Expression Estimation
McDermaid, A., Chen, X., Zhang, Y., Wang, C., Gu, S., Xie, J., & Ma, Q. (2018) · Frontiers in Genetics · doi:10.3389/fgene.2018.00313 · Cited by 31
“RNA-Seq is a revolutionary high-throughput process that allows researchers to observe the genetic makeup of a particular sample (Wang et al., 2009; Garber et al., 2011; Ozsolak and Milos, 2011) and can assist in determination of regulatory mechanisms and transcription unit prediction (Chou et al.”
Background
Multi-tissue transcriptomes of caecilian amphibians highlight incomplete knowledge of vertebrate gene families
Torres‐Sánchez, M., Creevey, C. J., Kornobis, É., Gower, D. J., Wilkinson, M., & Mauro, D. S. (2018) · DNA Research · doi:10.1093/dnares/dsy034 · Cited by 29
“Investigation of transcriptomes can allow the identification of functional elements of genomes, reveal molecular constituents of cells and tissues, help understand organismal development and disease,(6) and has the potential to uncover the role of tissue-specific evolution in biological diversity.(7) Having entered th…”
Background
Introduction: The Importance of Eggplant
Chapman, M. A. (2019) · Compendium of plant genomes · doi:10.1007/978-3-319-99208-2_1 · Cited by 30
CircMarker: a fast and accurate algorithm for circular RNA detection
Li, X., Chu, C., Pei, J., Măndoiu, I., & Wu, Y. (2018) · BMC Genomics · doi:10.1186/s12864-018-4926-0 · Cited by 30
“Recent studies show that sometimes circular RNA may be generated during transcription [14].”
Background
Expression and clinical significance of circular RNAs related to immunity and inflammation in patients with rheumatoid arthritis
Wen, J., Liu, J., Wang, X., & Wang, J. (2021) · International Immunopharmacology · doi:10.1016/j.intimp.2021.107366 · Cited by 30
Similarity-driven multi-view embeddings from high-dimensional biomedical data
Avants, B., Tustison, N. J., & Stone, J. R. (2021) · Nature Computational Science · doi:10.1038/s43588-021-00029-8 · Cited by 27
Prospects and challenges of cancer systems medicine: from genes to disease networks
Karimi, M. R., Karimi, A. H., Abolmaali, S., Sadeghi, M., & Schmitz, U. (2021) · Briefings in Bioinformatics · doi:10.1093/bib/bbab343 · Cited by 33
“The study of alternative splicing and novel splicing events [79, 80], variant calling [81, 82] and fusion transcript detection [83] are some of the other D ow naded rom http/academ ic.”
Background
Comparative transcriptomic analysis of the regulation of flowering in temperate and tropical lotus (Nelumbo nucifera) by RNA ‐Seq
Yang, M., Zhu, L., Xu, L., et al. (2014) · Annals of Applied Biology · doi:10.1111/aab.12119 · Cited by 30
Influence of temperature on thyroid hormone signaling and endocrine disruptor action in Rana (Lithobates) catesbeiana tadpoles
Hammond, S. A., Veldhoen, N., & Helbing, C. C. (2015) · General and Comparative Endocrinology · doi:10.1016/j.ygcen.2014.12.001 · Cited by 33
Plant and bacterial systems biology as platform for plant synthetic bio(techno)logy
Zurbriggen, M. D., Moor, A., & Weber, W. (2012) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2012.01.014 · Cited by 30
Agricultural applications of insect ecological genomics
Poelchau, M. F., Coates, B. S., Childers, C. P., de León, A. Á. P., Evans, J. D., Hackett, K. J., & Shoemaker, D. (2015) · Current Opinion in Insect Science · doi:10.1016/j.cois.2015.12.002 · Cited by 29
High E6 Gene Expression Predicts for Distant Metastasis and Poor Survival in Patients With HPV-Positive Oropharyngeal Squamous Cell Carcinoma
Khwaja, S. S., Baker, C. M., Haynes, W., et al. (2016) · International Journal of Radiation Oncology*Biology*Physics · doi:10.1016/j.ijrobp.2016.03.001 · Cited by 30
De novo sequencing and assembly of Azadirachta indica fruit transcriptome
Krishnan, N. M., Pattnaik, S., Deepak, S., Hariharan, A. K., Gaur, P., Chaudhary, R., Jain, P., Vaidyanathan, S., Krishna, P., & Panda, B. (2011) · Cited by 30
Identification and analysis of genes expressed in the Ustilago maydis dikaryon: uncovering a novel class of pathogenesis genes
Morrison, E. N., Donaldson, M. E., & Saville, B. J. (2012) · Canadian Journal of Plant Pathology · doi:10.1080/07060661.2012.697077 · Cited by 30
“The dikaryon cDNA library was normalized during its creation in an attempt to maximize gene identification (Ozsolak & Milos, 2011).”
Generative adversarial networks applied to gene expression analysis: An interdisciplinary perspective
Ai, X., Smith, M. C., & Feltus, F. A. (2023) · Computational and Systems Oncology · doi:10.1002/cso2.1050 · Cited by 30
Computational approaches for isoform detection and estimation: good and bad news
Angelini, C., Canditiis, D. D., & Feis, I. D. (2014) · BMC Bioinformatics · doi:10.1186/1471-2105-15-135 · Cited by 28
“To date, RNA-seq experiments have been successfully used in a wide spectrum of researches, offering tremendous benefits with respect to those previous approaches, such as microarrays, and also creatingmany challenges from both experimental and data analysis perspective [12].”
Method
Functional networks of aging markers in the glomeruli of IgA nephropathy: a new therapeutic opportunity
Jiang, H., Liang, L., Qin, J., Lu, Y., Li, B., Wang, Y., Lin, C., Zhou, Q., Feng, S., Yip, S. H., et al. (2016) · Oncotarget · doi:10.18632/oncotarget.9033 · Cited by 29
“RNA-Sequencing techniques [31-33] provide a good way to verify this hypothesis at a low cost.”
Background
Multiplex single‐cell quantification of rare RNA transcripts from protoplasts in a model plant system
Kadam, U. S., Schulz, B., & Irudayaraj, J. (2017) · The Plant Journal · doi:10.1111/tpj.13537 · Cited by 29
Analysis of global gene expression profiles during the flowering initiation process of Lilium × formolongi
Li, Y., Zhang, M., Zhang, M., & Jia, G. (2017) · Plant Molecular Biology · doi:10.1007/s11103-017-0612-x · Cited by 28
“Whole transcriptome sequencing using next-generation RNA sequencing (RNA-seq) technology is a convenient and rapid means to study gene expression at the wholegenome level and defines putative gene functions (Jain 2011; Ozsolak and Milos 2011).”
Method
Next-Generation Transcriptome Assembly: Strategies and Performance Analaysis
Voshall, A., & Moriyama, E. N. (2018) · InTech eBooks · doi:10.5772/intechopen.73497 · Cited by 29
“strains/species for both model and non-model organisms [1, 2].”
Background
Guidelines for RNA-seq projects: applications and opportunities in non-model decapod crustacean species
Nguyen, T. V., Jung, H., Rotllant, G., Hurwood, D. A., Mather, P. B., & Ventura, T. (2018) · Hydrobiologia · doi:10.1007/s10750-018-3682-0 · Cited by 29
Transcriptome Analysis Identified Genes for Growth and Omega-3/-6 Ratio in Saline Tilapia
Lin, G., Thevasagayam, N. M., Wan, Z., Ye, B. Q., & Yue, G. H. (2019) · Frontiers in Genetics · doi:10.3389/fgene.2019.00244 · Cited by 29
“Global gene expression and variation in transcription activity occurs across the genome and the number and level of transcript isoforms is not always known (Costa et al., 2010; Ozsolak and Milos, 2011; Pelechano et al., 2013).”
Background

Authors: Ozsolak, F., & Milos, P. M.
Affiliations: Helicos Biosciences (United States) (US)
Corresponding author: Ozsolak, Fatih
Milos, Patrice M.
Venue: Nature Reviews Genetics
Publisher: Springer Science and Business Media LLC
Published: 2010-12-30
Volume: 12
Issue: 2
Pages: 87-98
ISSN: 1471-0056 (ISSN-L)
1471-0064
DOI: 10.1038/nrg2934
PMC: PMC3031867
OpenAlex: W2060842569
Semantic Scholar: cf2841f590819568ab156862c2506ab633493747
Cited by: 2,135
References: 105
Citations by year: 2026: 52 · 2025: 108 · 2024: 113 · 2023: 98 · 2022: 131 · 2021: 152 · 2020: 146 · 2019: 134 · 2018: 141 · 2017: 173 · 2016: 163 · 2015: 169 · 2014: 169 · 2013: 168 · 2012: 152
FWCI: 10.78
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: www.springer.com/tdm (tdm)
Locations: Nature Reviews Genetics (publishedVersion)
PubMed (publishedVersion)
PubMed Central (acceptedVersion)
Funders: National Human Genome Research Institute (R01HG005230, R44HG005279)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords: RNA sequencing, transcriptome, direct RNA sequencing, RNA quantification, strand-specific RNA-seq, fusion gene detection, small RNA, alternative splicing
MeSH terms: Animals, Humans, RNA, Transcription, Genetic, Alternative Splicing, Sequence Analysis, RNA

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