Computational and analytical framework for small RNA profiling by high-throughput sequencing

  1. Noah Fahlgren1,2,
  2. Christopher M. Sullivan1,2,
  3. Kristin D. Kasschau1,2,
  4. Elisabeth J. Chapman1,2,3,
  5. Jason S. Cumbie1,2,
  6. Taiowa A. Montgomery1,2,
  7. Sunny D. Gilbert1,2,
  8. Mark Dasenko1,
  9. Tyler W.H. Backman1,2,
  10. Scott A. Givan1,2 and
  11. James C. Carrington1,2
  1. 1Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon 97331, USA
  2. 2Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA

    Abstract

    The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.

    Keywords:

    Keywords

    Footnotes

    • ↵3 Present address: Division of Biology, University of California at San Diego, La Jolla, CA 92093, USA.

    • Reprint requests to: James C. Carrington, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA; and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA; e-mail: carrington{at}cgrb.oregonstate.edu; fax: (541) 737-3045.

    • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1473809.

      • Received November 19, 2008.
      • Accepted January 23, 2009.
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    1. Published in Advance March 23, 2009, doi: 10.1261/rna.1473809 RNA 15: 992-1002 Copyright © 2009 RNA Society

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