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Rational experiment design for sequencing-based RNA structure mapping

Aviran, Sharon and Pachter, Lior (2014) Rational experiment design for sequencing-based RNA structure mapping. RNA, 20 (12). pp. 1864-1877. ISSN 1355-8382. PMCID PMC4238353. https://resolver.caltech.edu/CaltechAUTHORS:20170303-134607827

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1261/rna.043844.113DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238353/PubMed CentralArticle
ORCID:
AuthorORCID
Pachter, Lior0000-0002-9164-6231
Additional Information:© 2014 Aviran and Pachter; Published by Cold Spring Harbor Laboratory Press for the RNA Society. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. Received December 8, 2013. Accepted September 7, 2014. We thank Yiliang Ding, Julius Lucks, Shujun Luo, Stefanie Mortimer, and Silvi Rouskin for many discussions and for clarifications on the methods they developed. This work is supported by National Institutes of Health (NIH) grants R00 HG006860 to S.A. and R01 HG006129 to L.P.
Funders:
Funding AgencyGrant Number
NIHR00 HG006860
NIHR01 HG006129
Subject Keywords:next-generation sequencing, RNA structure, structure mapping, genomic big data, high-throughput genomics
Issue or Number:12
PubMed Central ID:PMC4238353
Record Number:CaltechAUTHORS:20170303-134607827
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170303-134607827
Official Citation:Rational experiment design for sequencing-based RNA structure mapping Sharon Aviran and Lior Pachter RNA December 2014 20: 1864-1877; Published in Advance October 20, 2014, doi:10.1261/rna.043844.113
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:74709
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:03 Mar 2017 23:54
Last Modified:27 Apr 2020 22:54

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