Fig 1: (A) An example of data output from the ddPCR multiplex assay for absolute quantification of PTPN11 mutations (PTPN11-1 for E76K variant and PTPN11-2 for the G503V variant) in canine HS. Exemplary two-dimensional cluster plots from QuantaSoft software (version 1.7.4, Bio-Rad, freely available after registration: https://www.bio-rad.com/fr-fr/sku/1864011-quantasoft-software-regulatory-edition) in which PTPN11-1mut (left) or PTPN11-2mut (right) are plotted against the PTPN11-1 wild-type (wt) and PTPN11-2wt respectively. The droplets form clusters that should arrange orthogonally to each other and represent the PTPN11mut/PTPN11wt negative (double-negative droplets, grey), PTPN11mut positive (blue), PTPN11wt positive (green), and PTPN11mut/PTPN11wt positive samples (double-positive droplets, orange). (B) Flowchart summarizing the results of the ddPCR assay targeting the PTPN11 mutations (E76K and G503V) in the tumor and plasma samples from 49 dogs with HS. cfDNA: cell-free DNA. (C) Parameters of the test for the detection of the PTPN11 mutation in the plasma of dogs with HS. PPV positive predictive value, NPV negative predictive value.