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RNA sequencing: advances, challenges and opportunities
Open accessAbstract
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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“RNA deep-sequencing (RNA-seq) provides a powerful tool to decipher global gene expression patterns far beyond the limitations of microarrays, including an unprecedented capability to discover novel genes, alternative transcript variants, chimeric transcripts, and expressed sequence variants as well as allele-specific expression [8–12].”
From Comparison of RNA-seq and microarray-based models for clinical endpoint prediction (2015) · cites this paper as [10]
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Chepelev, I., Wei, G., Tang, Q., & Zhao, K. (2009) · Nucleic Acids Research · doi:10.1093/nar/gkp507 · Cited by 182 · cited 1×
“The potential suitability of mRNA-seq data for the identification of nucleotide variations has been demonstrated recently by several studie”
MethodDigital transcriptome profiling using selective hexamer priming for cDNA synthesis
Armour, C. D., Castle, J. C., Chen, R., Babak, T., Loerch, P., Jackson, S., Shah, J. K., Dey, J., Rohl, C. A., Johnson, J. M., et al. (2009) · Nature Methods · doi:10.1038/nmeth.1360 · Cited by 176 · cited 1×
“First, the RNA-seq signal across transcripts tends to show non-uniformity of coverage, which may be a result of biases introduced during various steps, such as priming with random hexamer”
MethodDNA-directed DNA Polymerase Activity in Oncogenic RNA Viruses
SPIEGELMAN, S., BURNY, A., DAS, M. R., KEYDAR, J., SCHLOM, J., TRAVNICEK, M., & WATSON, K. (1970) · Nature · doi:10.1038/2271029a0 · Cited by 170 · cited 1×
“In addition, during first-strand cDNA synthesis, spurious second-strand cDNA artefacts can be introduced, owing to the DNA-dependent DNA polymerase (DDDP) activities of reverse transcriptase”
BackgroundReplication of individual DNA molecules under electronic control using a protein nanopore
Olasagasti, F., Lieberman, K. R., Benner, S., Cherf, G. M., Dahl, J. M., Deamer, D. W., & Akeson, M. A. (2010) · Nature Nanotechnology · doi:10.1038/nnano.2010.177 · Cited by 167 · cited 1×
“Examination of splicing patterns and transcript connectivity in an unbiased and genome-wide manner requires full-length transcript sequences to be obtained, which may be enabled in the future by emerging technologie”
A paired-end sequencing strategy to map the complex landscape of transcription initiation
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Prediction of alternative isoforms from exon expression levels in RNA-Seq experiments
Richard, H., Schulz, M. H., Sultan, M., Nürnberger, A., Schrinner, S., Balzereit, D., Dagand, E., Rasche, A., Lehrach, H., Vingron, M., et al. (2010) · Nucleic Acids Research · doi:10.1093/nar/gkq041 · Cited by 160 · cited 1×
“By counting the number of reads mapping to each exon and spanning each splice junction, these approaches also allowed the splice efficiency of each junction to be determined and the levels of distinct isoforms to be quantifie”
Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human
Zhang, K., Li, J. B., Gao, Y., Egli, D. M., Xie, B., Deng, J., Li, Z., Lee, J., Aach, J., LeProust, E. M., et al. (2009) · Nature Methods · doi:10.1038/nmeth.1357 · Cited by 159 · cited 1×
“Targeted RNA-seq approaches have been used to detect fusion transcripts, allele-specific expression, mutations and RNA-editing events in a subset of transcript”
MethodLinking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan
Plessy, C., Bertin, N., Takahashi, H., Simone, R., Salimullah, M., Lassmann, T., Vitezic, M., Severin, J., Olivarius, S., Lazarević, D., et al. (2010) · Nature Methods · doi:10.1038/nmeth.1470 · Cited by 154
Screening the human exome: a comparison of whole genome and whole transcriptome sequencing
Cirulli, E. T., Singh, A., Shianna, K. V., Ge, D., Smith, J. P., Maia, J. M., Heinzen, E. L., Goedert, J. J., & Goldstein, D. B. (2010) · Genome biology · doi:10.1186/gb-2010-11-5-r57 · Cited by 151 · cited 1×
“The potential suitability of mRNA-seq data for the identification of nucleotide variations has been demonstrated recently by several studie”
Genome-wide detection and analysis of hippocampus core promoters using DeepCAGEPreprint
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Virtual terminator nucleotides for next-generation DNA sequencing
Bowers, J., Mitchell, J., Beer, E., Buzby, P. R., Causey, M., Efcavitch, J. W., Jarosz, M. V., Krzymańska-Olejnik, E., Kung, L. A., Lipson, D., et al. (2009) · Nature Methods · doi:10.1038/nmeth.1354 · Cited by 139
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FRT-seq: amplification-free, strand-specific transcriptome sequencing
Mamanova, L., Andrews, R., James, K., Sheridan, E. M., Ellis, P. D., Langford, C. F., Ost, T. W. B., Collins, J. E., & Turner, D. (2010) · Nature Methods · doi:10.1038/nmeth.1417 · Cited by 134 · cited 1×
“The first involves the ligation of adaptors in a predetermined orientation to the ends of RNAs or to first-strand cDNA molecule”
BackgroundReplicase activity of purified recombinant protein P2 of double‐stranded RNA bacteriophage φ6
Makeyev, E. V., & Bamford, D. H. (2000) · The EMBO Journal · doi:10.1093/emboj/19.1.124 · Cited by 128
Linear mRNA Amplification from as Little as 5 ng Total RNA for Global gene Expression Analysis
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Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line
Zhao, Q., Caballero, O. L., Levy, S., Stevenson, B. J., Iseli, C., SOUZA, S. J. D., Galante, P. A. F., Busam, D., Leversha, M. A., Chadalavada, K., et al. (2009) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0812945106 · Cited by 120 · cited 1×
“However, RNA-seq may be preferable for identifying events that produce aberrant RNA species and therefore have a higher likelihood of being functional or causal in biological or disease setting”
BackgroundChromatin profiling by directly sequencing small quantities of immunoprecipitated DNA
Goren, A., Ozsolak, F., Shoresh, N., Ku, M., Adli, M., Hart, C., Gymrek, M., Zuk, O., Regev, A., Milos, P. M., et al. (2009) · Nature Methods · doi:10.1038/nmeth.1404 · Cited by 119 · cited 1×
“Third, the in-solution or on-surface amplification step included in some of these approaches may introduce additional artefacts — for example, in the form of GC biases and duplicate read”
BackgroundSelf-citationGlobal analysis of trans -splicing in Drosophila
McManus, C. J., Duff, M. O., Eipper-Mains, J. E., & Graveley, B. R. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1007586107 · Cited by 110 · cited 1×
“Furthermore, RNA-seq data include chimeric cDNA artefacts that are generated by template switching during reverse transcription and amplificatio”
MethodPolyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells
Shcherbik, N., Wang, M., Lapik, Y. R., Srivastava, L., & Pestov, D. G. (2010) · EMBO Reports · doi:10.1038/embor.2009.271 · Cited by 109 · cited 1×
“Polyadenylation of transcripts also takes place during transcript degradation steps, and thus poly(A) + -enrichment steps may also enrich for RNA degradation products of RNA polymerase I transcripts and other RNA”
BackgroundCharacterizing the mouse ES cell transcriptome with Illumina sequencing
Rosenkranz, R., Borodina, T., Lehrach, H., & Himmelbauer, H. (2008) · Genomics · doi:10.1016/j.ygeno.2008.05.011 · Cited by 106
New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism
Kapranov, P., Ozsolak, F., Kim, S. W., Foissac, S., Lipson, D., Hart, C., Roels, S., Borel, C., Antonarakis, S. E., Monaghan, A. P., et al. (2010) · Nature · doi:10.1038/nature09190 · Cited by 98 · cited 1×
“Because NGS sample preparation strategies for 'longer' RNAs (>200 nucleotides) are not suitable for sRNAs, such as reverse transcription with random priming (because this way of priming cDNA synthesis from short RNA species yields even shorter cDNA species that are not long enough for efficient alignment), modified pr…”
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“It has recently become clear that, although the NGS-based sRNA-profiling approaches can be used for differential expression analyses, the number of reads obtained per sRNA does not necessarily correlate with their actual abundanc”
BackgroundGlobal and unbiased detection of splice junctions from RNA-seq data
Ameur, A., Wetterbom, A., Feuk, L., & Gyllensten, U. B. (2010) · Genome biology · doi:10.1186/gb-2010-11-3-r34 · Cited by 94 · cited 1×
“As a result, it is now possible to search for splicing patterns without a requirement for prior knowledge of transcript annotation”
BackgroundEnhanced Generation of Induced Pluripotent Stem Cells from a Subpopulation of Human Fibroblasts
Byrne, J., Nguyen, H. N., & Pera, R. A. R. (2009) · PLoS ONE · doi:10.1371/journal.pone.0007118 · Cited by 75 · cited 1×
“This approach may be useful for the determination of the expression levels of a subset of transcripts across cells of interes”
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“, which can confound sense versus antisense transcript determinatio”
BackgroundWhen good transcripts go bad: artifactual RT‐PCR ‘splicing’ and genome analysis
Roy, S. W., & Irimia, M. (2008) · BioEssays · doi:10.1002/bies.20749 · Cited by 60 · cited 1×
“Another limitation imposed by cDNA synthesis is template switchin”
BackgroundDigital transcriptome profiling from attomole-level RNA samplesPreprint
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“A second approach is the direct sequencing of the first-strand cDNA products that are generated, either in solutio”
BackgroundSelf-citationAmplification-free digital gene expression profiling from minute cell quantities
Ozsolak, F., Ting, D. T., Wittner, B. S., Brannigan, B. W., Paul, S., Bardeesy, N. M., Ramaswamy, S., Milos, P. M., & Haber, D. A. (2010) · Nature Methods · doi:10.1038/nmeth.1480 · Cited by 59
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“This approach may be useful for the determination of the expression levels of a subset of transcripts across cells of interes”
BackgroundReverse Transcriptase Adds Nontemplated Nucleotides to cDNAs During 5′-RACE and Primer Extension
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“Another limitation imposed by cDNA synthesis is template switchin”
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McCarthy, D. J., Chen, Y., & Smyth, G. K. (2012) · Nucleic Acids Research · doi:10.1093/nar/gks042 · Cited by 5,195
Multi-omics approaches to disease
Hasin, Y., Seldin, M., & Lusis, A. (2017) · Genome Biology · doi:10.1186/s13059-017-1215-1 · Cited by 2,953
“Thus, transcriptomics, employing either expression arrays or RNA-Seq (Box 1), has proven to be particularly useful for identifying causal genes at GWAS loci [16, 22–24].”
BackgroundInfluentialGenome-wide genetic marker discovery and genotyping using next-generation sequencing
Davey, J. W., Hohenlohe, P. A., Etter, P. D., Boone, J. Q., Catchen, J., & Blaxter, M. (2011) · Nature Reviews Genetics · doi:10.1038/nrg3012 · Cited by 2,489
Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples
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“There are various approaches to solve that problem (Jiang and Wong 2009; Wang et al. 2009; Ozsolak and Milos 2011).”
BackgroundA census of human RNA-binding proteins
Gerstberger, S., Hafner, M., & Tuschl, T. (2014) · Nature Reviews Genetics · doi:10.1038/nrg3813 · Cited by 2,032
“, 2014), small and long RNA-sequencing (Jan et al., 2011; Ozsolak and Milos, 2011; Wang et al., 2009), or 3’-end sequencing methods profiling of alternative polyadenylation sites and poly(A) tail lengths (Chang et al.”
Background
Metadata
- Authors
- Ozsolak, F., & Milos, P. M.
- Affiliations
- Helicos Biosciences (United States) (US)
- Corresponding author
- Ozsolak, Fatih
- Milos, Patrice M.
- Venue
- Nature Reviews Genetics
- Published
- 2010-12-30
- Volume
- 12
- Issue
- 2
- Pages
- 87-98
- ISSN
- 1471-0056 (ISSN-L)
- 1471-0064
- 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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