RNA Structure Characterization from High-Throughput Chemical Mapping Experiments

Monday, February 13, 2012 - 3:45pm - 4:45pm
Keller 3-180
Sharon Aviran (University of California, Berkeley)
New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. This highlights a growing need to develop technologies that enable rapid and accurate characterization of structural features within complex RNA populations. Yet, available structure characterization techniques that are reliable are also vastly limited by technological constraints, while the accuracy of popular computational methods is generally poor. These limitations thus pose a major barrier to the comprehensive determination of structure from sequence and thereby to the development of mechanistic understanding of transcriptome dynamics. To address this need, we have recently developed a high-throughput structure characterization technique, called SHAPE-Seq, which simultaneously measures quantitative, single nucleotide-resolution, secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) chemical mapping with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline. Previous bioinformatics methods, in contrast, are laborious, heuristic, and expert-based, and thus prohibit high-throughput chemical mapping.

In this talk, I will review recent developments in experimental RNA structure characterization as well as advances in sequencing technologies. I will then describe the SHAPE-Seq technique, focusing on its automated data analysis method, which relies on a novel probabilistic model of a SHAPE-Seq experiment, adjoined by a rigorous maximum likelihood estimation framework. I will demonstrate the accuracy and simplicity of our approach as well as its applicability to a general class of chemical mapping techniques and to more traditional SHAPE experiments that use capillary electrophoresis to identify and quantify primer extension products.

This is joint work with Lior Pachter, Julius Lucks, Stefanie Mortimer, Shujun Luo, Cole Trapnell, Gary Schroth, Jennifer Doudna and Adam Arkin.
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