Profiling of Short RNAs Using Helicos Single-Molecule Sequencing

  • Philipp Kapranov
  • Fatih Ozsolak
  • Patrice M. Milos

Abstract

The importance of short (<200 nt) RNAs in cell biogenesis has been well documented. These short RNAs include crucial classes of molecules such as transfer RNAs, small nuclear RNA, microRNAs, and many others (reviewed in Storz et al., Annu Rev Biochem 74:199–217, 2005; Ghildiyal and Zamore, Nat Rev Genet 10:94–108, 2009). Furthermore, the realm of functional RNAs that fall within this size range is growing to include less well-characterized RNAs such as short RNAs found at the promoters and 3′ termini of genes (Affymetrix ENCODE Transcriptome Project et al., Nature 457:1028–1032, 2009; Davis and Ares, Proc Natl Acad Sci USA 103:3262–3267, 2006; Kapranov et al., Science 316:1484–1488, 2007; Taft et al., Nat Genet 41:572–578, 2009; Kapranov et al., Nature 466:642–646, 2010), short RNAs involved in paramutation (Rassoulzadegan et al., Nature 441:469–474, 2006), and others (reviewed in Kawaji and Hayashizaki, PLoS Genet 4:e22, 2008). Discovery and accurate quantification of these RNA molecules, less than 200 bases in size, is thus an important and also challenging aspect of understanding the full repertoire of cellular and extracellular RNAs. Here, we describe the strategies and procedures we developed to profile short RNA species using single-molecule sequencing (SMS) and the advantages SMS offers.

Key words

Single-molecule sequencing Short RNAs Promoter-associate short RNAs Polyad­enylated short RNAs 

References

  1. 1.
    Storz, G., Altuvia, S., and Wassarman, K. M. (2005) An abundance of RNA regulators. Annu Rev Biochem 74, 199–217.Google Scholar
  2. 2.
    Ghildiyal, M., and Zamore, P. D. (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10, 94–108.Google Scholar
  3. 3.
    Affymetrix, ENCODE, Transcriptome, Project;, Cold, Spring, et al. (2009) Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature 457, 1028–32.Google Scholar
  4. 4.
    Davis, C. A., and Ares, M., Jr. (2006) Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 103, 3262–7.Google Scholar
  5. 5.
    Kapranov, P., Cheng, J., Dike, S., Nix, D. A., Duttagupta, R., Willingham, A. T., et al. (2007) RNA maps reveal new RNA classes and a possible function for pervasive. transcription Science 316, 1484–8.Google Scholar
  6. 6.
    Taft, R. J., Glazov, E. A., Cloonan, N., Simons, C., Stephen, S., Faulkner, G. J., et al. (2009) Tiny RNAs associated with transcription start sites in animals. Nat Genet 41, 572–8.Google Scholar
  7. 7.
    Kapranov, P., Ozsolak, F., Kim, S. W., Foissac, F., Lipson, D., Hart, C., et al. (2010) Novel class of human RNAs associated with gene termini suggests an uncharacterized RNA copying mechanism. Nature 466, 642–6.Google Scholar
  8. 8.
    Rassoulzadegan, M., Grandjean, V., Gounon, P., Vincent, S., Gillot, I., and Cuzin, F. (2006) RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 441, 469–74.Google Scholar
  9. 9.
    Kawaji, H., and Hayashizaki, Y. (2008) Exploration of small RNAs. PLoS Genet 4, e22.Google Scholar
  10. 10.
    Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105, 10513–8.Google Scholar
  11. 11.
    Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., et al. (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18, 997–1006.Google Scholar
  12. 12.
    Sambrook, J., and Russell, D. W. (2001) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  13. 13.
    Giladi, E., Healy, J., Myers, G., Hart, C., Kapranov, P., Lipson, D., et al. (2010) Error tolerant indexing and alignment of short reads with covering template families. J. Comput. Biol. 17, 1397–411.Google Scholar
  14. 14.
    Harris, T. D., Buzby, P. R., Babcock, H., Beer, E., Bowers, J., Braslavsky, I., et al. (2008) Single-molecule DNA sequencing of a viral genome. Science 320, 106–9.Google Scholar
  15. 15.
    Lipson, D., Raz, T., Kieu, A., Jones, D. R., Giladi, E., Thayer, E., et al. (2009) Quantification of the yeast transcriptome by single-molecule sequencing. Nat Biotechnol 27, 652–8.Google Scholar
  16. 16.
    Li, H., and Durbin, R. (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–60.Google Scholar
  17. 17.
    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.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Philipp Kapranov
    • 1
  • Fatih Ozsolak
    • 1
  • Patrice M. Milos
    • 1
  1. 1.Helicos BioSciences CorporationCambridgeUSA

Personalised recommendations