Comparative ctDNA Analysis in Tumor, CSF, and Plasma

gDNA extracted from tumor tissue and cells, and cfDNA extracted from CSF and plasma, was pre-amplified at CN using Q5 hot start high-fidelity master mix (New England Biolabs), and at NU using SsoAdvanced PreAmp Supermix (Biorad), with 50 nmol/L each of forward and reverse primer. Pre-amplification at CN was performed using Assay A primers (Supplementary Fig. 1) in ABI 2720 thermocycler: 98 °C for 3 min; nine cycles of 98 °C for 10 s, 58 °C for 3 min, 72 °C for 30 s; and an extension of 72 °C for 2 min. Product was diluted 1:5 with TE buffer (pH 8.0). Pre-amplification at NU was performed on the BioRad T100 thermocycler using the following conditions: 95 °C for 3 min, 10 cycles of 95 °C for 15 s, annealing temperature (58 °C) for 4 min. The pre-amplified product was diluted 1:5 with molecular grade water. At CN, 0.025 ng gDNA from DMG-51-T was used as a positive control per a previously established institutional protocol^{17 (link),19 (link)}. 2 ng of tumor gDNA was used for ddPCR analysis of patient-matched tumor, CSF and plasma/serum specimens. Where applicable, starting cfDNA aliquots were speed-vacuum concentrated from 100 µL to 10.5–11 µL prior to pre-amplification. Assay A primers were used for ctDNA pre-amplification of all samples at CN, while Assay C primers were used for PCR pre-amplification at NU^{18 (link)}.