Abstract
Methods for in-depth characterization of transcriptomes and quantification of transcript levels have emerged as valuable tools for understanding cellular physiology and human disease biology, and have begun to be utilized in various clinical diagnostic applications. Today, current methods utilized by the scientific community typically require RNA to be converted to cDNA prior to comprehensive measurements. However, this cDNA conversion process has been shown to introduce many biases and artifacts that interfere with the proper characterization and quantitation of transcripts. We have developed a direct RNA sequencing (DRS) approach, in which, unlike other technologies, RNA is sequenced directly without prior conversion to cDNA. The benefits of DRS include the ability to use minute quantities (e.g. on the order of several femtomoles) of RNA with minimal sample preparation, the ability to analyze short RNAs which pose unique challenges for analysis using cDNA-based approaches, and the ability to perform these analyses in a low-cost and high-throughput manner. Here, we describe the strategies and procedures we employ to prepare various RNA species for analysis with DRS.
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References
Fodor, S. P., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T., and Solas, D. (1991) Light-directed, spatially addressable parallel chemical synthesis, Science 251, 767–  773.
Lennon, G. G., and Lehrach, H. (1991) Hybridization analyses of arrayed cDNA libraries, Trends Genet 7, 314  –317.
Shalon, D., Smith, S. J., and Brown, P. O. (1996) A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization, Genome Res 6, 639–  645.
Southern, E. M., Maskos, U., and Elder, J. K. (1992) Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models, Genomics 13, 1008–1017.
Bennett, S. T., Barnes, C., Cox, A., Davies, L., and Brown, C. (2005) Toward the 1,000 dollars human genome, Pharmacogenomics 6, 373–382.
Deamer, D. W., and Branton, D. (2002) Characterization of nucleic acids by nanopore analysis, Acc Chem Res 35, 817–825.
Levene, M. J., Korlach, J., Turner, S. W., Foquet, M., Craighead, H. G., and Webb, W. W. (2003) Zero-mode waveguides for single-molecule analysis at high concentrations, Science 299, 682–  686.
Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., Berka, J., Braverman, M. S., Chen, Y. J., Chen, Z., Dewell, S. B., Du, L., Fierro, J. M., Gomes, X. V., Godwin, B. C., He, W., Helgesen, S., Ho, C. H., Irzyk, G. P., Jando, S. C., Alenquer, M. L., Jarvie, T. P., Jirage, K. B., Kim, J. B., Knight, J. R., Lanza, J. R., Leamon, J. H., Lefkowitz, S. M., Lei, M., Li, J., Lohman, K. L., Lu, H., Makhijani, V. B., McDade, K. E., McKenna, M. P., Myers, E. W., Nickerson, E., Nobile, J. R., Plant, R., Puc, B. P., Ronan, M. T., Roth, G. T., Sarkis, G. J., Simons, J. F., Simpson, J. W., Srinivasan, M., Tartaro, K. R., Tomasz, A., Vogt, K. A., Volkmer, G. A., Wang, S. H., Wang, Y., Weiner, M. P., Yu, P., Begley, R. F., and Rothberg, J. M. (2005) Genome sequencing in microfabricated high-density picolitre reactors, Nature 437, 376–380.
Shendure, J., Porreca, G. J., Reppas, N. B., Lin, X., McCutcheon, J. P., Rosenbaum, A. M., Wang, M. D., Zhang, K., Mitra, R. D., and Church, G. M. (2005) Accurate multiplex polony sequencing of an evolved bacterial genome, Science 309, 1728–1732.
Valouev, A., Ichikawa, J., Tonthat, T., Stuart, J., Ranade, S., Peckham, H., Zeng, K., Malek, J. A., Costa, G., McKernan, K., Sidow, A., Fire, A., and Johnson, S. M. (2008) A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning, Genome Res 18, 1051–1063.
Kapranov, P., Willingham, A. T., and Gingeras, T. R. (2007) Genome-wide transcription and the implications for genomic organization, Nat Rev Genet 8, 413–  423.
Denoeud, F., Aury, J. M., Da Silva, C., Noel, B., Rogier, O., Delledonne, M., Morgante, M., Valle, G., Wincker, P., Scarpelli, C., Jaillon, O., and Artiguenave, F. (2008) Annotating genomes with massive-scale RNA sequencing, Genome Biol 9, R175.
Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M., and Gilad, Y. (2008) RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays, Genome Res 18, 1509  –1517.
Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., and Wold, B. (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq, Nat Methods 5, 621–  628.
Nagalakshmi, U., Wang, Z., Waern, K., Shou, C., Raha, D., Gerstein, M., and Snyder, M. (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing, Science 320, 1344  –1349.
Sultan, M., Schulz, M. H., Richard, H., Magen, A., Klingenhoff, A., Scherf, M., Seifert, M., Borodina, T., Soldatov, A., Parkhomchuk, D., Schmidt, D., O’Keeffe, S., Haas, S., Vingron, M., Lehrach, H., and Yaspo, M. L. (2008) A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome, Science 321, 956  –960.
Wilhelm, B. T., Marguerat, S., Watt, S., Schubert, F., Wood, V., Goodhead, I., Penkett, C. J., Rogers, J., and Bahler, J. (2008) Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution, Nature 453, 1239  –1243.
Gubler, U. (1987) Second-strand cDNA synthesis: classical method, Methods Enzymol 152, 325  –329.
Gubler, U. (1987) Second-strand cDNA synthesis: mRNA fragments as primers, Methods Enzymol 152, 330  –  335.
Spiegelman, S., Burny, A., Das, M. R., Keydar, J., Schlom, J., Travnicek, M., and Watson, K. (1970) DNA-directed DNA polymerase activity in oncogenic RNA viruses, Nature 227, 1029  –1031.
Wu, J. Q., Du, J., Rozowsky, J., Zhang, Z., Urban, A. E., Euskirchen, G., Weissman, S., Gerstein, M., and Snyder, M. (2008) Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome, Genome Biol 9, R3.
Cocquet, J., Chong, A., Zhang, G., and Veitia, R. A. (2006) Reverse transcriptase template switching and false alternative transcripts, Genomics 88, 127–131.
Mader, R. M., Schmidt, W. M., Sedivy, R., Rizovski, B., Braun, J., Kalipciyan, M., Exner, M., Steger, G. G., and Mueller, M. W. (2001) 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.
Roy, S. W., and Irimia, M. (2008) When good transcripts go bad: artifactual RT-PCR ‘splicing’ and genome analysis, Bioessays 30, 601–  605.
Roy, S. W., and Irimia, M. (2008) Intron mis-splicing: no alternative?, Genome Biol 9, 208.
Haddad, F., Qin, A. X., Bodell, P. W., Zhang, L. Y., Guo, H., Giger, J. M., and Baldwin, K. M. (2006) Regulation of antisense RNA expression during cardiac MHC gene switching in response to pressure overload, Am J Physiol Heart Circ Physiol 290, H2351–2361.
Haddad, F., Qin, A. X., Giger, J. M., Guo, H., and Baldwin, K. M. (2007) Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR, BMC Biotechnol 7, 21.
Roberts, J. D., Preston, B. D., Johnston, L. A., Soni, A., Loeb, L. A., and Kunkel, T. A. (1989) Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro, Mol Cell Biol 9, 469–  476.
Varadaraj, K., and Skinner, D. M. (1994) Denaturants or cosolvents improve the specificity of PCR amplification of a G  +  C-rich DNA using genetically engineered DNA polymerases, Gene 140, 1–  5.
Cloonan, N., Forrest, A. R., Kolle, G., Gardiner, B. B., Faulkner, G. J., Brown, M. K., Taylor, D. F., Steptoe, A. L., Wani, S., Bethel, G., Robertson, A. J., Perkins, A. C., Bruce, S. J., Lee, C. C., Ranade, S. S., Peckham, H. E., Manning, J. M., McKernan, K. J., and Grimmond, S. M. (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing, Nat Methods 5, 613–  619.
Lister, R., O’Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., and Ecker, J. R. (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis, Cell 133, 523–  536.
Ozsolak, F., Platt, A. R., Jones, D. R., Reifenberger, J. G., Sass, L. E., McInerney, P., Thompson, J. F., Bowers, J., Jarosz, M., and Milos, P. M. (2009) Direct RNA sequencing, Nature 461, 814–  818.
Li, H., and Durbin, R. (2009) Fast and accurate short read alignment with Burrows-Wheeler transform, Bioinformatics 25, 1754  –1760.
Rumble, S. M., Lacroute, P., Dalca, A. V., Fiume, M., Sidow, A., and Brudno, M. (2009) SHRiMP: accurate mapping of short color-space reads, PLoS Comput Biol 5, e1000386.
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We thank our colleagues at the Helicos BioSciences Corporation for technical assistance and discussions.
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Ozsolak, F., Milos, P.M. (2011). Transcriptome Profiling Using Single-Molecule Direct RNA Sequencing. In: Kwon, Y., Ricke, S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology, vol 733. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-089-8_4
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DOI: https://doi.org/10.1007/978-1-61779-089-8_4
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