Identification of cancer-related mutations in human pluripotent stem cells using RNA-seq analysis

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. Researchers from the Hebrew University of Jerusalem 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, they 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.

Schematic representation of the pipeline for identification of cancer-related
mutations in hPSCs from RNA-seq data

Fig. 2

a, Colored boxes represent the main stages of the procedure; the tools used in each stage are between parentheses in italics. b, Schematic representation of the pipeline where each step is represented by a box; input and output files are between the steps in italics.

Availability – this pipeline is designed to work in high throughput and is available as a software container at https://github.com/elyadlezmi/RNA2CM.

Lezmi E, Benvenisty N. (2021) Identification of cancer-related mutations in human pluripotent stem cells using RNA-seq analysis. Nat Protoc [Epub ahead of print]. [abstract].

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