Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organization, North Ryde, Australia; Department of Biomedical Sciences, Macquarie Park, Australia.
John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia; Department of Biomedical Sciences, Macquarie Park, Australia.
CRISPR J. 2021 Apr;4(2):243-252. doi: 10.1089/crispr.2020.0068.
The increased development of functionally diverse and highly specialized genome editors has created the need for comparative analytics tools that are able to profile the mutational outcomes, particularly rare and complex outcomes, to assess the editor's applicability to different domains. To address this need, we have developed Generalizable On-target activity ANAlyzer (GOANA), a high-throughput web-based software for determining editing efficiency and cataloguing rare outcomes from next-generation sequencing data. GOANA calculates mutation frequency and outcomes relative to a supplied control sample. It is scalable to thousands of target sites across the entire genome and is 4,000% faster than CRISPResso2. Mutations are reported on a "per-read" level rather than individually, enabling the identification of co-occurring mutations. GOANA is editor agnostic and can be applied to data generated from any targeted editing experiment, including base editors. Requiring only that control and treated reads are aligned to the same reference, GOANA can handle data from any library preparation method, including pooled amplicon and whole-genome sequencing. As a proof of principle, we analyze two large data sets of CRISPR-Cas9 and CRISPR-Cas12a editing, demonstrating the power of GOANA and highlighting several key differences between the two enzymes. GOANA is available for use at https://gt-scan.csiro.au/goana/ and as a command line tool from https://github.com/BauerLab/GOANA.
功能多样化和高度专业化的基因组编辑工具不断发展，这就需要开发能够分析突变结果（尤其是罕见和复杂的结果）的比较分析工具，以评估编辑工具在不同领域的适用性。为满足这一需求，我们开发了可推广的靶向活性分析器（GOANA），这是一种用于确定编辑效率并从下一代测序数据中编目罕见结果的高通量基于网络的软件。GOANA 相对于提供的对照样本计算突变频率和结果。它可扩展到整个基因组中的数千个靶标位点，比 CRISPResso2 快 4000%。突变以“每读”为单位报告，而不是单独报告，从而能够识别同时发生的突变。GOANA 不依赖于编辑工具，可以应用于任何靶向编辑实验生成的数据，包括碱基编辑器。GOANA 只需控制和处理的读数与同一参考对齐，就可以处理来自任何文库制备方法的数据，包括池扩增和全基因组测序。作为原理验证，我们分析了两个 CRISPR-Cas9 和 CRISPR-Cas12a 编辑的大型数据集，展示了 GOANA 的强大功能，并突出了两种酶之间的几个关键差异。GOANA 可在 https://gt-scan.csiro.au/goana/ 上使用，并可从 https://github.com/BauerLab/GOANA 获得命令行工具。
