title: Computational and analytical framework for small RNA profiling by high-throughput sequencing.
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.
authors.affiliation: Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon 97331, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Fahlgren
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Sullivan
authors.forename: Kristin D
authors.initials: KD
authors.lastname: Kasschau
authors.forename: Elisabeth J
authors.initials: EJ
authors.lastname: Chapman
authors.forename: Jason S
authors.initials: JS
authors.lastname: Cumbie
authors.forename: Taiowa A
authors.initials: TA
authors.lastname: Montgomery
authors.forename: Sunny D
authors.initials: SD
authors.lastname: Gilbert
authors.forename: Mark
authors.initials: M
authors.lastname: Dasenko
authors.forename: Tyler W H
authors.initials: TW
authors.lastname: Backman
authors.forename: Scott A
authors.initials: SA
authors.lastname: Givan
authors.forename: James C
authors.initials: JC
authors.lastname: Carrington
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D019295:Computational Biology; D020869:Gene Expression Profiling; D018749:RNA, Plant / Q000235:genetics*; D017423:Sequence Analysis, RNA
chemical_list: D018749:RNA, Plant