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Transcriptome Profiling Using Single-Molecule Direct RNA Sequencing

Part of the Methods in Molecular Biology book series (MIMB,volume 733)

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.

Key words

  • RNA sequencing
  • Single-molecule sequencing
  • Transcriptome profiling
  • Polyadenylation site mapping

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Acknowledgments

We thank our colleagues at the Helicos BioSciences Corporation for technical assistance and discussions.

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Correspondence to Patrice M. Milos .

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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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