Chromothripsis is a frequent form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or multiple chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumour, as well as changes in response to treatment. Here we analyse single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma and neural stem cells (n = 9). We reconstruct the order of somatic events, identify early alterations likely linked to chromothripsis and depict the contribution of chromothripsis to malignancy. We characterise subclonal variation of chromothripsis and its effects on extrachromosomal circular DNA, cancer drivers and putatively druggable targets. Furthermore, we highlight the causative role and the fitness consequences of specific rearrangements in neural progenitors.We thank Peter Lichter and Aurelio Teleman for discussions, Frauke Devens, Michaela Hergt, Brigitte Schoell and Katharina Bauer for technical support, the Sequencing and the Microarray units of the Genomics and Proteomics Core Facility (DKFZ), the EMBL Sequencing Facility, the DKFZ FACS Core Facility and the DKFZ Imaging Facility. David Pellman and his team are acknowledged for kindly sharing their published single-cell DNA sequencing data as a reference dataset. Florian Markowetz and his team are acknowledged for sharing scAbsolute and for support in using it. Thomas Weber and Jan Korbel are acknowledged for their advice regarding strand-seq data analysis. Axel Benner is acknowledged for support with statistical analyses. Daniel Haag is acknowledged for kindly sharing neural stem cells. P.S. was supported by the Heidelberg-Mannheim Life Science Alliance. D.R.G. was supported with personal grants by the German Academic Scholarship Foundation (Studienstiftung des Deutschen Volkes) and the Mildred Scheel Doctoral Fellowship programme of the German Cancer Aid (Deutsche Krebshilfe). K.W.P. acknowledges funding by the German Childhood Cancer Foundation (DKS2021.02) and the Federal Ministry of Education and Research (01GM2205A). R.G.P. holds a fellowship from Grant IHMC22/00007 funded by the Instituto de Salud Carlos III (ISCIII). The vast majority of the experimental work in this study was supported by grants to A.E. from the DFG, the Wilhelm Sander Foundation and the Fritz Thyssen Foundation. H.S. was supported by the German Federal Ministry of Education and Research (031L069A).Peer Reviewed"Article signat per 26 autors/es: Petr Smirnov, Moritz J. Przybilla, Milena Simovic-Lorenz, R. Gonzalo Parra, Hana Susak, Manasi Ratnaparkhe, John KL. Wong, Verena Körber, Jan-Philipp Mallm, George Philippos, Martin Sill, Thorsten Kolb, Rithu Kumar, Nicola Casiraghi, Konstantin Okonechnikov, David R. Ghasemi, Kendra Korinna Maaß, Kristian W. Pajtler, Anna Jauch, Andrey Korshunov, Thomas Höfer, Marc Zapatka, Stefan M. Pfister, Wolfgang Huber, Oliver Stegle & Aurélie Ernst"Postprint (published version
