RNA sequencing: advances, challenges and opportunities (original) (raw)

• Birney, E. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).
  Article CAS PubMed Google Scholar
• Berretta, J. & Morillon, A. Pervasive transcription constitutes a new level of eukaryotic genome regulation. EMBO Rep. 10, 973–982 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Kapranov, P., Willingham, A. T. & Gingeras, T. R. Genome-wide transcription and the implications for genomic organization. Nature Rev. Genet. 8, 413–423 (2007).
  Article CAS PubMed Google Scholar
• Metzker, M. L. Sequencing technologies — the next generation. Nature Rev. Genet. 11, 31–46 (2010). This Review provides a comprehensive overview of currently available and in-development NGS technologies.
  Article CAS PubMed Google Scholar
• Wang, Z., Gerstein, M. & Snyder, M. RNA-seq: a revolutionary tool for transcriptomics. Nature Rev. Genet. 10, 57–63 (2009).
  Article CAS PubMed Google Scholar
• van Vliet, A. H. Next generation sequencing of microbial transcriptomes: challenges and opportunities. FEMS Microbiol. Lett. 302, 1–7 (2010).
  Article CAS PubMed Google Scholar
• Ozsolak, F. et al. Direct RNA sequencing. Nature 461, 814–818 (2009). The first technology for high-throughput direct sequencing of RNA molecules without prior reverse transcription.
  Article CAS PubMed Google Scholar
• Carninci, P. et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nature Genet. 38, 626–635 (2006).
  Article CAS PubMed Google Scholar
• Shiraki, T. et al. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc. Natl Acad. Sci. USA 100, 15776–15781 (2003).
  Article CAS PubMed PubMed Central Google Scholar
• Valen, E. et al. Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE. Genome Res. 19, 255–265 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Ni, T. et al. A paired-end sequencing strategy to map the complex landscape of transcription initiation. Nature Methods 7, 521–527 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Plessy, C. et al. Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nature Methods 7, 528–534 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Marson, A. et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134, 521–533 (2008).
  Article CAS PubMed PubMed Central Google Scholar
• Ozsolak, F. et al. Chromatin structure analyses identify miRNA promoters. Genes Dev. 22, 3172–3183 (2008).
  Article CAS PubMed PubMed Central Google Scholar
• Affymetrix/Cold Spring Harbor Laboratory ENCODE Transcriptome Project. Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature 457, 1028–1032 (2009). This paper raises the possibility of 5′-cap addition during post-transcriptional processing steps.
• Faghihi, M. A. & Wahlestedt, C. Regulatory roles of natural antisense transcripts. Nature Rev. Mol. Cell Biol. 10, 637–643 (2009). An excellent review of the literature on sense and antisense transcription.
  Article CAS Google Scholar
• Gubler, U. Second-strand cDNA synthesis: mRNA fragments as primers. Meth. Enzymol. 152, 330–335 (1987).
  Article CAS Google Scholar
• Perocchi, F., Xu, Z., Clauder-Munster, S. & Steinmetz, L. M. Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D. Nucleic Acids Res. 35, e128 (2007).
  Article PubMed PubMed Central CAS Google Scholar
• Spiegelman, S. et al. DNA-directed DNA polymerase activity in oncogenic RNA viruses. Nature 227, 1029–1031 (1970).
  Article CAS PubMed Google Scholar
• Wu, J. Q. et al. Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome. Genome Biol. 9, R3 (2008).
  Article PubMed PubMed Central CAS Google Scholar
• Cloonan, N. et al. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nature Methods 5, 613–619 (2008).
  Article CAS PubMed Google Scholar
• Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).
  Article CAS PubMed PubMed Central Google Scholar
• Mamanova, L. et al. FRT-seq: amplification-free, strand-specific transcriptome sequencing. Nature Methods 7, 130–132 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Lipson, D. et al. Quantification of the yeast transcriptome by single-molecule sequencing. Nature Biotechnol. 27, 652–658 (2009).
  Article CAS Google Scholar
• Ozsolak, F. et al. Digital transcriptome profiling from attomole-level RNA samples. Genome Res. 20, 519–525 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Ozsolak, F. et al. Amplification-free digital gene expression profiling from minute cell quantities. Nature Methods 7, 619–621 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• He, Y., Vogelstein, B., Velculescu, V. E., Papadopoulos, N. & Kinzler, K. W. The antisense transcriptomes of human cells. Science 322, 1855–1857 (2008).
  Article CAS PubMed PubMed Central Google Scholar
• Parkhomchuk, D. et al. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 37, e123 (2009).
  Article PubMed PubMed Central CAS Google Scholar
• Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Levin, J. Z. et al. Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nature Methods 7, 709–715 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Faulhammer, D., Lipton, R. J. & Landweber, L. F. Fidelity of enzymatic ligation for DNA computing. J. Comput. Biol. 7, 839–848 (2000).
  Article CAS PubMed Google Scholar
• Housby, J. N. & Southern, E. M. Fidelity of DNA ligation: a novel experimental approach based on the polymerisation of libraries of oligonucleotides. Nucleic Acids Res. 26, 4259–4266 (1998).
  Article CAS PubMed PubMed Central Google Scholar
• Dohm, J. C., Lottaz, C., Borodina, T. & Himmelbauer, H. Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 36, e105 (2008).
  Article PubMed PubMed Central CAS Google Scholar
• Goren, A. et al. Chromatin profiling by directly sequencing small quantities of immunoprecipitated DNA. Nature Methods 7, 47–49 (2010).
  Article CAS PubMed Google Scholar
• Kozarewa, I. et al. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nature Methods 6, 291–295 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Nilsen, T. W. & Graveley, B. R. Expansion of the eukaryotic proteome by alternative splicing. Nature 463, 457–463 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Wang, G. S. & Cooper, T. A. Splicing in disease: disruption of the splicing code and the decoding machinery. Nature Rev. Genet. 8, 749–761 (2007).
  Article CAS PubMed Google Scholar
• Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nature Methods 5, 621–628 (2008).
  Article CAS PubMed Google Scholar
• Sultan, M. et al. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 321, 956–960 (2008).
  Article CAS PubMed Google Scholar
• Tang, F. et al. mRNA-seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377–382 (2009).
  Article CAS PubMed Google Scholar
• Wang, E. T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).
  Article CAS PubMed PubMed Central Google Scholar
• Carninci, P. Is sequencing enlightenment ending the dark age of the transcriptome? Nature Methods 6, 711–713 (2009).
  Article CAS PubMed Google Scholar
• Jiang, H. & Wong, W. H. Statistical inferences for isoform expression in RNA-seq. Bioinformatics 25, 1026–1032 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Richard, H. et al. Prediction of alternative isoforms from exon expression levels in RNA-seq experiments. Nucleic Acids Res. 38, e112 (2010).
  Article PubMed PubMed Central CAS Google Scholar
• Ameur, A., Wetterbom, A., Feuk, L. & Gyllensten, U. Global and unbiased detection of splice junctions from RNA-seq data. Genome Biol. 11, R34 (2010).
  Article PubMed PubMed Central CAS Google Scholar
• Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).
  Article CAS PubMed Google Scholar
• Olasagasti, F. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nature Nanotech. 5, 798–806 (2010).
  Article CAS Google Scholar
• Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene fusions on cancer causation. Nature Rev. Cancer 7, 233–245 (2007).
  Article CAS Google Scholar
• Korbel, J. O. et al. Paired-end mapping reveals extensive structural variation in the human genome. Science 318, 420–426 (2007).
  Article CAS PubMed PubMed Central Google Scholar
• Maher, C. A. et al. Transcriptome sequencing to detect gene fusions in cancer. Nature 458, 97–101 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Zhao, Q. et al. Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line. Proc. Natl Acad. Sci. USA 106, 1886–1891 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Li, H., Wang, J., Mor, G. & Sklar, J. A neoplastic gene fusion mimics trans-splicing of RNAs in normal human cells. Science 321, 1357–1361 (2008).
  Article CAS PubMed Google Scholar
• Maher, C. A. et al. Chimeric transcript discovery by paired-end transcriptome sequencing. Proc. Natl Acad. Sci. USA 106, 12353–12358 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Berger, M. F. et al. Integrative analysis of the melanoma transcriptome. Genome Res. 20, 413–427 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Palanisamy, N. et al. Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma. Nature Med. 16, 793–798 (2010).
  Article CAS PubMed Google Scholar
• McManus, C. J., Duff, M. O., Eipper-Mains, J. & Graveley, B. R. Global analysis of trans-splicing in Drosophila. Proc. Natl Acad. Sci. USA 107, 12975–12979 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Garber, K. Fixing the front end. Nature Biotech. 26, 1101–1104 (2008).
  Article CAS Google Scholar
• Chepelev, I., Wei, G., Tang, Q. & Zhao, K. Detection of single nucleotide variations in expressed exons of the human genome using RNA-seq. Nucleic Acids Res. 37, e106 (2009).
  Article PubMed PubMed Central CAS Google Scholar
• Cirulli, E. T. et al. Screening the human exome: a comparison of whole genome and whole transcriptome sequencing. Genome Biol. 11 (2010).
  Article PubMed PubMed Central CAS Google Scholar
• Shah, S. P. et al. Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461, 809–813 (2009).
  Article CAS PubMed Google Scholar
• Levin, J. Z. et al. Targeted next-generation sequencing of a cancer transcriptome enhances detection of sequence variants and novel fusion transcripts. Genome Biol. 10, R115 (2009).
  Article PubMed PubMed Central CAS Google Scholar
• Li, J. B. et al. Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science 324, 1210–1213 (2009).
  CAS PubMed Google Scholar
• Zhang, K. et al. Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human. Nature Methods 6, 613–618 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nature Rev. Genet. 10, 94–108 (2009).
  Article CAS PubMed Google Scholar
• Rajagopalan, R., Vaucheret, H., Trejo, J. & Bartel, D. P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20, 3407–3425 (2006).
  Article CAS PubMed PubMed Central Google Scholar
• Ruby, J. G. et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207 (2006).
  Article CAS PubMed Google Scholar
• Seila, A. C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008).
  Article CAS PubMed PubMed Central Google Scholar
• Taft, R. J. et al. Tiny RNAs associated with transcription start sites in animals. Nature Genet. 41, 572–578 (2009).
  Article CAS PubMed Google Scholar
• Berezikov, E. et al. Diversity of microRNAs in human and chimpanzee brain. Nature Genet. 38, 1375–1377 (2006).
  Article CAS PubMed Google Scholar
• Kapranov, P. et al. New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism. Nature 466, 642–646 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862 (2001).
  Article CAS PubMed Google Scholar
• Kawaji, H. & Hayashizaki, Y. Exploration of small RNAs. PLoS Genet. 4, e22 (2008).
  Article PubMed PubMed Central CAS Google Scholar
• Linsen, S. E. et al. Limitations and possibilities of small RNA digital gene expression profiling. Nature Methods 6, 474–476 (2009). The authors describe the difficulties associated with the analysis and quantification of short RNA species using current NGS platforms.
  Article CAS PubMed Google Scholar
• Cocquet, J., Chong, A., Zhang, G. & Veitia, R. A. Reverse transcriptase template switching and false alternative transcripts. Genomics 88, 127–131 (2006).
  Article CAS PubMed Google Scholar
• Mader, R. M. et al. Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA. J. Lab. Clin. Med. 137, 422–428 (2001).
  Article CAS PubMed Google Scholar
• Roy, S. W. & Irimia, M. When good transcripts go bad: artifactual RT-PCR 'splicing' and genome analysis. Bioessays 30, 601–605 (2008).
  Article CAS PubMed Google Scholar
• Chen, D. & Patton, J. T. Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5′-RACE and primer extension. Biotechniques 30, 574–582 (2001).
  Article CAS PubMed Google Scholar
• Roberts, J. D. et al. Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro. Mol. Cell. Biol. 9, 469–476 (1989).
  Article CAS PubMed PubMed Central Google Scholar
• Armour, C. D. et al. Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nature Methods 6, 647–649 (2009).
  Article CAS PubMed Google Scholar
• Hansen, K. D., Brenner, S. E. & Dudoit, S. Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic Acids Res. 38, e131 (2010).
  Article PubMed PubMed Central CAS Google Scholar
• Rosenkranz, R., Borodina, T., Lehrach, H. & Himmelbauer, H. Characterizing the mouse ES cell transcriptome with Illumina sequencing. Genomics 92, 187–194 (2008).
  Article CAS PubMed Google Scholar
• Oshlack, A. & Wakefield, M. J. Transcript length bias in RNA-seq data confounds systems biology. Biol. Direct 4, 14 (2009).
  Article PubMed PubMed Central CAS Google Scholar
• Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010).
  Article PubMed PubMed Central CAS Google Scholar
• Li, B., Ruotti, V., Stewart, R. M., Thomson, J. A. & Dewey, C. N. RNA-seq gene expression estimation with read mapping uncertainty. Bioinformatics 26, 493–500 (2010).
  Article PubMed CAS Google Scholar
• Guttman, M. et al. Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nature Biotech. 28, 503–510 (2010).
  Article CAS Google Scholar
• Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotech. 28, 511–515 (2010).
  Article CAS Google Scholar
• Shcherbik, N., Wang, M., Lapik, Y. R., Srivastava, L. & Pestov, D. G. Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells. EMBO Rep. 11, 106–111 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Makeyev, E. V. & Bamford, D. H. Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6. EMBO J. 19, 124–133 (2000).
  Article CAS PubMed PubMed Central Google Scholar
• Gurumurthy, S. et al. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature 468, 659–63 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Ozsolak, F. et al. Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation. Cell 143, 1018–1029 (2010).
  Article CAS PubMed PubMed Central Google Scholar
• Simone, N. L., Bonner, R. F., Gillespie, J. W., Emmert-Buck, M. R. & Liotta, L. A. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet. 14, 272–276 (1998).
  Article CAS PubMed Google Scholar
• Marcy, Y. et al. Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl Acad. Sci. USA 104, 11889–11894 (2007).
  Article CAS PubMed PubMed Central Google Scholar
• Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B. & Riggs, A. D. Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813 (1989).
  Article CAS PubMed Google Scholar
• Dean, F. B. et al. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl Acad. Sci. USA 99, 5261–5266 (2002).
  Article CAS PubMed PubMed Central Google Scholar
• Dafforn, A. et al. Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis. Biotechniques 37, 854–857 (2004).
  Article CAS PubMed Google Scholar
• Eberwine, J. et al. Analysis of gene expression in single live neurons. Proc. Natl Acad. Sci. USA 89, 3010–3014 (1992).
  Article CAS PubMed PubMed Central Google Scholar
• Nygaard, V. & Hovig, E. Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling. Nucleic Acids Res. 34, 996–1014 (2006). This review provides a good overview of the current low-quantity RNA applications and the complications associated with them.
  Article CAS PubMed PubMed Central Google Scholar
• Geiss, G. K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotech. 26, 317–325 (2008).
  Article CAS Google Scholar
• Amit, I. et al. Unbiased reconstruction of a mammalian transcriptional network mediating pathogen responses. Science 326, 257–263 (2009).
  Article CAS PubMed PubMed Central Google Scholar
• Byrne, J. A., Nguyen, H. N. & Reijo Pera, R. A. Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts. PLoS ONE 4, e7118 (2009).
  Article PubMed PubMed Central CAS Google Scholar
• Helzer, K. T. et al. Circulating tumor cells are transcriptionally similar to the primary tumor in a murine prostate model. Cancer Res. 69, 7860–7866 (2009).
  Article CAS PubMed Google Scholar
• Lo, Y. M. et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nature Med. 13, 218–223 (2007). This paper describes the quantification of extracellular circulating RNA in mother's plasma during pregnancy to detect fetal aneuploidy.
  Article CAS PubMed Google Scholar
• Bowers, J. et al. Virtual terminator nucleotides for next-generation DNA sequencing. Nature Methods 6, 593–595 (2009).
  Article CAS PubMed PubMed Central Google Scholar