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

Lucca L M Derks; Anaïs J C N van Leeuwen; Alexander S Steemers; Laurianne Trabut; Markus J van Roosmalen; Vera M Poort; Rico Hagelaar; Mark Verheul; Sjors Middelkamp; Ruben van Boxtel

doi:10.1016/j.xpro.2024.103499

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

STAR Protoc. 2024 Dec 20;6(1):103499. doi: 10.1016/j.xpro.2024.103499. Online ahead of print.

Affiliations

¹ Princess Máxima Center for Pediatric Oncology, Utrecht 3584 CS, the Netherlands; Oncode Institute, Utrecht 3521 AL, the Netherlands.
² Oncode Institute, Utrecht 3521 AL, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Utrecht 3584 CG, the Netherlands.
³ Princess Máxima Center for Pediatric Oncology, Utrecht 3584 CS, the Netherlands; Oncode Institute, Utrecht 3521 AL, the Netherlands. Electronic address: r.vanboxtel@prinsesmaximacentrum.nl.

Abstract

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.

Keywords: bioinformatics; cancer; molecular biology; sequence analysis; sequencing.