Targeted amplicon sequencing of rhCMV (Macacine betaherpesvirus 3) gL loci: Urine from macaque HD79 in treatment group Control at week 8
Viral DNA was isolated from plasma using the High Pure Viral Nucleic Acid kit (Roche Life Science). Hypervariable regions 400bp in length within glycoprotein B and glycoprotein L were amplified in duplicate by a single round of PCR using 5-AAGTGCTCGAAGGGCTTCTC-3 and 5-TCTGGACATTGATCCGCTGG-3 for the gB region and 5-GCGCGGCACACATTATCTAC-3 and 5-GGTGAGTGCTGCTGTTTTGG-3 for the gL region. Overhang regions were conjugated to primer locus-specific sequences for subsequent Illumina index primer addition and sequencing. Approximately 10ng DNA was added as template to 50L of 1PCR mixture containing 100nM of each primer, 2mM MgCl2, 200M each of dNTP mix (Qiagen), 0.025U/L Phusion Taq polymerase. PCR conditions consisted of an initial 2min denaturation at 98C, followed by the minimum number of PCR cycles to achieve adequate amplification (98C for 10s, 65C for 30s, and 72C for 30s), and a final 72C extension for 10min. Products were purified using Agencourt AMPure XP beads (Beckman Coulter), then Illumina Nextera XT index primers were added by 15 cycles of amplification. The resultant PCR product was gel purified using ZR-96 Zymoclean Gel DNA Recovery Kit (Zymogen). The molar amount of each sample was normalized by qPCR using the KAPA library amplification kit (KAPA Biosystems). The library of individual amplicons was pooled together, diluted to an end concentration of 10pm, combined with 20% V3 PhiX (Illumina), then sequenced on Illumina Miseq using a 600 cycle V3 cartridge (Illumina).