title: Rational experiment design for sequencing-based RNA structure mapping.
abstract: Structure mapping is a classic experimental approach for determining nucleic acid structure that has gained renewed interest in recent years following advances in chemistry, genomics, and informatics. The approach encompasses numerous techniques that use different means to introduce nucleotide-level modifications in a structure-dependent manner. Modifications are assayed via cDNA fragment analysis, using electrophoresis or next-generation sequencing (NGS). The recent advent of NGS has dramatically increased the throughput, multiplexing capacity, and scope of RNA structure mapping assays, thereby opening new possibilities for genome-scale, de novo, and in vivo studies. From an informatics standpoint, NGS is more informative than prior technologies by virtue of delivering direct molecular measurements in the form of digital sequence counts. Motivated by these new capabilities, we introduce a novel model-based in silico approach for quantitative design of large-scale multiplexed NGS structure mapping assays, which takes advantage of the direct and digital nature of NGS readouts. We use it to characterize the relationship between controllable experimental parameters and the precision of mapping measurements. Our results highlight the complexity of these dependencies and shed light on relevant tradeoffs and pitfalls, which can be difficult to discern by intuition alone. We demonstrate our approach by quantitatively assessing the robustness of SHAPE-Seq measurements, obtained by multiplexing SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry in conjunction with NGS. We then utilize it to elucidate design considerations in advanced genome-wide approaches for probing the transcriptome, which recently obtained in vivo information using dimethyl sulfate (DMS) chemistry.
authors.affiliation: Biomedical Engineering Department and Genome Center, University of California at Davis, Davis, California 95616, USA saviran@ucdavis.edu.
authors.forename: Sharon
authors.initials: S
authors.lastname: Aviran
authors.affiliation: Center for Computational Biology and Departments of Molecular and Cell Biology and Mathematics, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Lior
authors.initials: L
authors.lastname: Pachter
mesh_terms: D019295:Computational Biology / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D009690:Nucleic Acid Conformation; D017423:Sequence Analysis, RNA; D059467:Transcriptome / Q000235:genetics*
chemical_list: D018076:DNA, Complementary
keywords: RNA structure; genomic big data; high-throughput genomics; next-generation sequencing; structure mapping