Rhesus CMV genomic sequencing; 001-101 amniotic fluid day 77
Based on viral load, we used minimum 1000 input copies for amplification and library preparation, where available. AF at 82 days PI had approximately 300 input copies, while plasma at 77 and 82 days PI had fewer than 100 input copies. Sample DNA was non-specifically amplified using multiple displacement amplification (MDA, Repli-g, Qiagen #150025) then purified and concentrated using Zymo Genomic DNA Clean and Concentrate kit (Zymo Research, D4011). Sample DNA was equally split across 96 wells, each containing a single primer pair to span the entire genome and PCR amplified using the pure PfuUltra II Fusion HS DNA Polymerase (Agilent #600674). Primer sequences are listed in Supplementary Data excel file. Amplicons were pooled together then purified using QIAprep spin mini prep kit (Qiagen #27106). Using 500 ng of purified DNA, library prep was performed using NEB FS DNA library prep kit (E7805) and dual-index Illumina adapters. Libraries were run on Illumina Nextseq 2000 platform using a 200-cycle cartridge.