Quantstudio 3d platform

The SEMA4A V78M mutation was quantitatively analysed with digital PCR (dPCR) on the QuantStudio 3D platform (Life Technologies). A Custom TaqMan SNP Genotyping Assay specific for the analysis of the V78M mutation was used and tested on a StepOne Plus instrument (Life Technologies) using the TaqMan Genotyping Master Mix (Life Technologies) according to the manufacturer's recommendations. For dPCR, 17.4 μl of Digital PCR Master Mix (2 × ) was mixed with 1.7 μl of the TaqMan assay and 60 ng of DNA to a final volume of 36 μl and subjected to two Digital PCR 20k Chips. The chips were thermally cycled in a two-step PCR using the GeneAmp PCR System 9700 (10 min 96 °C, followed by 44 cycles of 56 °C 2 min and 94 °C 30 s, final extension of 2 min 58 °C) and imaged in the QuantStudio 3D instrument. Raw data were analysed using the Relative Quantification module of the QuantStudio 3D AnalysisSuite Software. The confidence level was set to 95% and the desired precision value was 10%.

SCNAs of POLR1D and ERBB2 were analyzed using digital PCR (dPCR) and performed on the QuantStudio 3D platform (Life Technologies, Carlsbad, CA, USA). Pre-designed TaqMan assays specific for the detection of the copy number of POLR1D (Hs02926936_cn), ERBB2 (Hs00450668_cn), and a reference assay (TERT; 4403315) were purchased from Thermo Fisher. For dPCR, a total amount of 3–5 ng plasma DNA was used as input and samples were run in duplicate using the QuantStudio™ 3D Digital PCR 20 K Chip Kit v2 and a QuantStudio 3D instrument (Life Technologies, Carlsbad, CA, USA). Raw data were analyzed using the relative quantification module of the QuantStudio 3D Analysis Suite Software, including a Poisson correction. The confidence level was set to 95%, and the desired precision value was 10%.