Abstract
RNA structure is a primary determinant of its function, and methods that merge chemical probing with next generation sequencing have created breakthroughs in the throughput and scale of RNA structure characterization. However, little work has been done to examine the effects of library preparation and sequencing on the measured chemical probe reactivities that encode RNA structural information. Here, we present the first analysis and optimization of these effects for selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly reproducible reactivity data over a wide range of RNA structural contexts with no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a ‘universal’ method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding the RNA sequence–structure relationship at the heart of some of life's most fundamental processes.
Highlights
RNAs play diverse functional roles in many natural cellular processes [1], and are being increasingly engineered to control these processes in many synthetic biology and biotechnology applications [2]
Several such techniques have been developed (selective 2 -hydroxyl acylation analyzed by primer extension sequencing (SHAPESeq) [6,7], DMS-Seq [9,10,13], MAP-Seq [8]) that each follow the same general protocol consisting of: (i) structuredependent modification of the RNA in vitro or in vivo; (ii) reverse transcription (RT) of the modified RNA into a cDNA pool whose length distribution reflects the location of modifications; (iii) sequencing library construction, involving the addition of platform-specific adapter sequences to the cDNA pool, optional amplification with polymerase chain reaction (PCR) and quality control assessment steps; (iv) sequencing of the library and (v) bioinformatic processing of sequencing reads and calculation of reactivity spectra for each RNA (Figure 1)
There are two distinct protocol steps associated with sequencing library preparation that distinguish SHAPE-Seq from SHAPE analyzed by capillary electrophoresis (Figure 1, Supplementary Figure S1): adapter ligation and PCR
Summary
RNAs play diverse functional roles in many natural cellular processes [1], and are being increasingly engineered to control these processes in many synthetic biology and biotechnology applications [2]. Because of the inherent multiplexing and enormous throughput offered by sequencing-based approaches, these techniques are providing some of the first ‘genome-wide’ snapshots of RNA structure [9,10]––effectively bringing RNA structural biology into the ‘omics’ era [11]. Of these techniques, those that favor chemical probing over nuclease digests show the most promise because of the inherent versatility [12], higher resolution and in vivo accessibility [9,10,13,14,15] of many chemical probes. Very little work has been done to evaluate the impact of these extra steps
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