Protocol for genome-wide analysis of somatic variants at single-cell resolution using primary template-directed DNA amplification

The study of somatic mutations in single cells provides insights into aging and carcinogenesis, which is complicated by the dependency on whole-genome amplification (WGA). Here, we describe a detailed workflow starting from single-cell isolation to WGA by primary template-directed amplification (PTA), sequencing, quality control, and downstream analyses. A machine learning approach, the PTA Analysis Toolkit (PTATO), is used to filter the hundreds to thousands of artificial variants induced by WGA from true mutations at high sensitivity and accuracy. For complete details on the use and execution of this protocol, please refer to Middelkamp et al.1 [Display omitted] •Isolation, whole-genome amplification, and sequencing of single cells•Removal of artificial variants from somatic mutations using a machine learning approach•Implementation of quality control steps in experimental and computational workflows Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The study of somatic mutations in single cells provides insights into aging and carcinogenesis, which is complicated by the dependency on whole-genome amplification (WGA). Here, we describe a detailed workflow starting from single-cell isolation to WGA by primary template-directed amplification (PTA), sequencing, quality control, and downstream analyses. A machine learning approach, the PTA Analysis Toolkit (PTATO), is used to filter the hundreds to thousands of artificial variants induced by WGA from true mutations at high sensitivity and accuracy.