Q5 hot start high fidelity master mix

We extracted HBV DNA from 200 ul of patient serum samples using a QIAamp MinElute Virus Spin Kit (QIAGEN, Hilden, Germany), following procedures provided by the manufacturer. A DNA fragment containing the preS1 region was specifically amplified using Q5 Hot Start High-Fidelity Master Mix (New England Biolabs, MA, USA) with the following primers: forward, 5′-AAGGTGGGAAACTTTACGGG-3′; reverse, 5′-TGACAWACTTTCCAATCAATAGG-3′. PCR conditions are as follows: 98°C for 30 s; 98°C for 10 s, 55°C for 20 s, and 72°C for 50 s for 35 cycles, and 72°C for 2 min (final extension). PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Invitrogen, CA, USA), and sequencing was performed on an ABI 3730XL DNA analyzer. The HBV sub-genomic fragments were submitted to the NCBI website to identify the genotype (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi). We aligned and compared the amplified HBV sequences with the HBV genotype B (GenBank accession no. D00330) or genotype C (GenBank accession no. AB033556) reference sequence to determine preS1 fragment variants.

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)}.

An initial preamplification reaction was run prior to ddPCR in the case of very low DNA concentration. cfDNA were pre-amplified using Q5 hot start high-fidelity master mix (New England Biolabs, Beverly, MA, USA) with forward and reverse primer pair for EGFRvIII and TERT C228T (same primers used for ctDNA analysis). Pre-amplification was performed with the Eppendorf Mastercycler: 98°C for 3 min; 12 cycles of 98°C for 30 s, 60°C for 1 min; a final extension of 72°C for 5 min, and 1 cycle at 4°C infinite. Preamplified products were directly used for further ddPCR reactions.