HiTRACE: high-throughput robust analysis for capillary electrophoresis

Abstract

We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on 13 datasets representing 4 different RNAs, 3 chemical modification strategies and up to 480 single mutant variants; the largest datasets each include 87 360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate datasets. Furthermore, while the smallest of these datasets required 7-10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest datasets herein was achieved in 3-12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

“…We obtained results that were the same, within estimated experimental error, upon varying folding solution conditions (Wilkinson et al 2006;Deigan et al 2009;Kladwang et al 2011b), acylating reagents (1M7 and NMIA) (Merino et al 2005;Mortimer and Weeks 2007), reverse transcription conditions (Mills and Kramer 1979), quantitation software (Yoon et al 2011;Karabiber et al 2013), normalization schemes (Deigan et al 2009), and RNAstructure modeling software versions ( Fig. 1D; Supplemental Figs.…”

“…The HiTRACE software package version 2.0 was used to analyze CE data (MATLAB toolbox is available at https://github.com/hitrace) (Yoon et al 2011), and a web server is also available at http:// hitrace.org (Kim et al 2013). Electrophoretic traces were aligned and baseline subtracted using linear and nonlinear alignment routines as previously described (Kim et al 2009).…”

“…One approach for probing large numbers of RNA molecules involves chemically modifying RNA and reading out these events via electrophoresis or deep sequencing (Mitra et al 2008;Lucks et al 2011;Pang et al 2011;Yoon et al 2011). Numerous reagents, including protein nucleases (Walczak et al 1996;Grover et al 2011;Siegfried et al 2011), alkylating chemicals such as dimethyl sulfate (DMS) (Wells et al 2000;Tijerina et al 2007;Cordero et al 2012a), and hydroxyl radicals (Adilakshmi et al 2006;Das et al 2008;Ding et al 2012), have been leveraged to modify or cleave RNA in a structure-dependent manner.…”

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

“…Extracting quantitative 4 reactivities for each nucleotide requires extensive multistep analytical signal processing. Diverse software tools have been developed to facilitate processing of electropherograms, including CAFA (Mitra et al 2008), ShapeFinder , HiTRACE (Yoon et al 2011), FAST (Pang et al 2011), and SHAPE-CE (Aviran et al 2011b). There is a critical balance to be struck between processing speed, pipeline simplicity, and degree of automation.…”

RNA structure through multidimensional chemical mapping

“…The proposal also motivated advances in high-throughput protocols for chemical mapping of these variants, replacing radioactive labeling of primers and slab gel electrophoresis with fluorescent readouts and capillary electrophoresis instruments developed for Sanger sequencing (Kladwang et al 2011a;Mitra et al 2008;Yoon et al 2011). These accelerations now allow M 2 measurements to be carried out and analyzed in 2 days, after the receipt of automatically designed primers for template assembly from commercial DNA companies Lee et al 2015;Tian et al 2015).…”