School of Electrical Engineering, Korea University, Seoul 136-713, Republic of Korea.
Bioinformatics. 2011 Jul 1;27(13):1798-805. doi: 10.1093/bioinformatics/btr277. Epub 2011 May 10.
Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment.
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.
核酸毛细管电泳（CE）是高通量基因组分析和大规模化学绘图的基础技术，可用于核酸结构推断。尽管 CE 基仪器广泛可用，但在利用其全部功能进行 RNA 和 DNA 结构、热力学和动力学的定量分析方面仍然存在挑战。特别是，可用分析工具的速度慢和自动化程度低，限制了涉及每个实验数百个 CE 图谱的新一代研究。
我们提出了一种称为高通量毛细管电泳的鲁棒分析的计算方法（HiTRACE），以实现大规模核酸 CE 分析的关键任务的自动化，包括迄今为止一直是最高通量实验的限速步骤的图谱对齐。我们展示了 HiTRACE 在 13 个数据集上的应用，这些数据集代表 4 种不同的 RNA、3 种化学修饰策略和多达 480 种单突变变体；最大的数据集每个都包含 87360 个条带。通过应用一系列鲁棒动态规划算法，HiTRACE 在对齐和拟合质量方面优于先前的工具，评估指标包括重复数据集之间定量条带强度之间的相关性。此外，尽管这些数据集中最小的数据集需要使用先前方法进行 7-10 小时的手动干预，但本文中甚至最大数据集的 HiTRACE 定量分析也在 3-12 分钟内完成。因此，HiTRACE 方法解决了在涉及数万条电泳条带的实验中有效且准确分析核酸结构的关键障碍。
