Design of primers for digital droplet PCR (ddPCR)

ddPCR assays were designed using the Bio-Rad Droplet Digital PCR Assays and tools available online for mutation (https://www.bio-rad.com/digital-assays/#/assays-create/mutation) or copy number determination (https://www.bio-rad.com/digital-assays/#/assays-create/cnd) to detect the PTPN11 mutations (E76K and G503V) or determine MDM2, TRPM7, and CFA 9 copy numbers, respectively. The region of CFA 9 was used as an internal control, as it was previously shown to have a high copy number stability in OMM^28,56. The Bio-Rad MIQE Context locations of primers and probes are shown in Table ​Table22.

For each dog with multicentric lymphoma examined for MRD, a PARR assay was performed on the tumor DNA as described above to isolate the clonal antigen receptor rearrangement. The PCR product was sequenced using a 3130 ABI sequencer with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequences overlapping the CDR3 (complementarity-determining region 3) were used to design clone-specific primers and FAM fluorescent probes for ddPCR analysis using the PrimerQuest Tool (Integrated DNA Technologies, Coralville, IA, USA). A region of CFA26 was used as the control gene, as it was previously found to be relatively stable in canine lymphomas⁵⁷ (Table ​(Table22).