Genomiphi v2 dna amplification kit

Total DNA was extracted from 3 × 20 ml of pure culture. The culture was centrifuged at 3200×g for 20 min at 4°C in 2 × 50 ml tubes, combined in a 1.5 ml tube, centrifuged at 10 000×g, and the supernatant eliminated. The cell pellet was homogenized in 600 µL of lysis buffer (5% CTAB, 0.7 M NaCl, 240 mM potassium phosphate buffer, pH 7.5, and 2% β-mercaptoethanol) in the presence of glass beads. A volume of 600 µL of phenol–chloroform–isoamyl (PCI, 25:24:1 v/v/v, pH 4.5) was added, vortexed for 1 min, and incubated at 65°C for 5 min. The sample was centrifuged at 11 000×g for 10 min at 4°C. The aqueous phase containing the DNA was transferred to a 1.5-ml tube, 400 µL of isoamyl chloroform (24:1 v/v) was added, vortexed, and centrifuged at 11 000×g for 5 min at 4°C. The aqueous was precipitated with 18% polyethylene glycol overnight, centrifuged at 15 000×g for 30 min at 4°C, purified, and eluted in Tris low ethylene diamine tetraacetic acid (EDTA) buffer (10 mM Tris Ultrapure, 0.1 mM EDTA, and pH 8.0). The DNA amplification was achieved using the GenomiPhi V2 DNA Amplification Kit (Cytiva), following the manufacturer’s instructions. The sequencing library was prepared according to the MGI Easy FS DNA library preparation kit and the sample was sequenced with 2 × 200 bp using a DNBSEQ-G400 high-throughput sequencer (MGI Technology).

Nucleic acids were extracted using the QIAamp MinElute Virus Spin kit (Qiagen, Germany) from 200 μL of concentrated viral fraction according to manufacturer’s guidelines. The viral DNA was used for whole genome multiple displacement amplification (MDA) using random hexamers with the illustra GenomiPhi V2 DNA amplification kit (Cytiva, USA) according to the manufacturer’s guidelines. The amplified DNA was then purified using the ethanol precipitation method [66 ]. Briefly, sodium acetate was added and mixed to the amplified sample to a final concentration of 0.3 M at pH 5.2. Two volumes of cold 100% molecular grade ethanol was added and incubated overnight at − 20 °C. After incubation, the sample was centrifuged at 15,000 g for 30 min and the supernatant was removed. One mL of 70% ethanol was then added and incubated at − 20 °C for 2 h before centrifuging at 15,000 g for 30 min to pellet DNA. The supernatant was discarded and the pellet air dried for 5 min before resuspension in sterile dH₂O.

DNA extraction from clinical samples was conducted according to [27 (link)] (with some modifications). Briefly, each biopsy was shaken on a 3D shaker for 3 h at 4 °C and centrifuged at 12,000× g for 10 min. The supernatant was filtered through a 0.22 μm pore membrane and loaded on a CsCl gradient (density layers of 1.7, 1.5, 1.35 and 1.15 g mL⁻¹) for virome separation. Cesium chloride centrifugation was performed at 62,000× g, at 4 °C for 22–24 h. The obtained virome was located between the density fractions of 1.5 and 1.35 g mL⁻¹ and withdrawn for the further procedure. Virome DNA isolation (Sherlock AX kit, A&A Biotechnology, Gdańsk, Poland) was carried out and samples with minimum DNA concentrations of 1 ng/µL were amplified (Genomiphi V2 DNA Amplification kit, Cytiva, Marlborough, MA, USA). To prepare sequencing libraries, Illumina DNA Prep with Nextera DNA CD indexes (Illumina, San Diego, CA, USA) was used. The virome DNA concentration was estimated using a Quantus Fluorometer with the QuantiFluor dsDNA system (Promega, Walldorf, Germany).