Divya Sivaraman

Human cytomegalovirus (HCMV) is a genetically complex virus that can transmit congenitally causing long-term health problems in infants. When HCMV is grown in tissue culture, adaptive mutations can develop that impact viral replication and tropism. Due to the adaptation of viruses’ growth in tissue culture, it is increasingly expected that next-generation sequencing will be used to validate the identity of stocks and strains during virology experiments. In this research project, HCMV obtained from a clinical sample was passaged on ARPE-19 cells, and we sequenced this new strain to compare its genome relative to the HCMV reference. The passaged virus was sequenced using the Illumina MiSeq in a 300bp, paired-end run format. GRACy software automates and integrates modules for read filtering, genome assembly, genome annotation, variant analysis, and data submission for analyzing HCMV sequence data. Using GRACy software, next-generation sequencing reads were assembled, annotated, and compared to the published genomes to fix gaps and assemble a single viral chromosome. To span the two gaps after assembly, primers were designed at the ends of the contigs and the gaps were closed by Sanger sequencing. Once the full-length viral genome was constructed, annotations transferred to the novel genome by GRACy were manually analyzed by comparison to previously-sequenced strains related to our isolate, confirming the location of splice sites and open reading frames. The project could be further developed in the future by sequencing both the parental urine sample plus the fibroblast strain to see whether there are any mutants.