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Circulating tumour cells (CTCs) are heterogenous and contain genetic information from the tumour of origin. They bear specific intra- and extra-cellular protein markers aiding in their detection. However, since these markers may be shared with other rare cells in the blood, only genetic testing can confirm their malignancy. Herein, we analyse different CTC subsets using single cell whole genome DNA sequencing to validate their malignant origin. We randomly selected putative CTCs identified by immunostaining that were isolated from 4 patients with high grade serous ovarian cancer (HGSOC) and one with benign cystadenoma. We specifically targeted CTCs positive for epithelial (CK/EpCAM pos ), mesenchymal (vimentin pos ), and pseudoendothelial (CK/EpCAM pos plus CD31 pos ) markers. We isolated these cells and performed whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for analysis of copy number alterations (CNA). Of the CK/EpCAM pos cells analysed from the HGSOC patients, 2 of 3 cells showed diverse chromosomal CNAs. However, the 4 pseudoendothelial cells (CK/EpCAM pos plus CD31 pos ) observed in the HGSOC cases did not carry any CNA. Lastly, two of the clusters of vimentin positive cells sequenced from those found in the benign cystadenoma case had CNA. Despite the low number of cells analysed, our results underscore the importance of genetic analysis of putative CTCs to confirm their neoplastic origin. In particular, it highlights the presence of a population of CK/EpCAM pos cells that are not tumour cells in patients with HGSOC, which otherwise would be counted as CTCs., (© 2023. The Author(s).)