Human pluripotent stem cells (hPSCs) are known to acquire genetic aberrations during in vitro propagation. In addition to recurrent chromosomal aberrations, it has recently been shown that these cells also gain point mutations in cancer-related genes, predominantly in TP53. The need for routine quality control of hPSCs is critical for both basic research and clinical applications. Here we discuss the relevance of detecting mutations for various hPSCs applications, and present a detailed protocol to identify cancer-related point mutations using data from RNA sequencing, an assay commonly performed during the growth and differentiation of hPSCs. In this protocol, we describe how to process and align the sequencing data, analyze it and conservatively interpret the results in order to generate an accurate estimation of mutations in tumor-related genes. This pipeline is designed to work in high throughput and is available as a software container at https://github.com/elyadlezmi/RNA2CM. The protocol requires minimal command-line skills and can be carried out in 1–2 d.

已知人类多能干细胞 (hPSC) 在体外繁殖过程中会获得遗传畸变。除了复发性染色体畸变外，最近显示这些细胞还在癌症相关基因中获得点突变，主要在 TP53 中。对 hPSC 进行常规质量控制的需求对于基础研究和临床应用都至关重要。在这里，我们讨论了检测各种 hPSC 应用的突变的相关性，并提出了一个详细的协议，以使用来自 RNA 测序的数据来识别癌症相关的点突变，这是一种在 hPSC 的生长和分化过程中通常进行的检测。在本协议中，我们描述了如何处理和对齐测序数据，对其进行分析并保守地解释结果，以便准确估计肿瘤相关基因的突变。该管道旨在以高通量工作，可在 https://github.com/elyadlezmi/RNA2CM 作为软件容器使用。该协议需要最少的命令行技能，并且可以在 1-2 d 内执行。