DNA sequencing of human cytomegalovirus genomes from formalin-fixed, paraffin-embedded tissues from congenital cytomegalovirus disease cases

Background: Congenital cytomegalovirus disease (cCMV) is a severe but uncommon condition. The role of variation in the causative virus (human cytomegalovirus, HCMV) in clinical outcome have to date depended on small sample numbers derived from fresh tissues. Extensive formalin-fixed, paraffin-embedded (FFPE) cCMV biorepositories established worldwide potentially provide much larger sample numbers for future investigations. However, there are no published reports of sequencing HCMV genomes from such material.Objective: To assess the feasibility of sequencing HCMV genomes from cCMV FFPE material.Study design: Sixteen FFPE samples of foetal kidney or placental tissue were processed from ten cCMV cases in foetuses or neonates. Two commercial kits for extracting DNA from FFPE material were evaluated, HCMV DNA was enriched in the extracts, and the samples were sequenced on the Illumina platform. The sequence read datasets were analysed by genotyping, genome assembly and variant calling using a published software pipeline.Results: Using either DNA extraction kit, genotyping a set of hypervariable HCMV genes was achieved for nine cases, and HCMV genomes were sequenced for five cases. Significant variation was noted at only one nucleotide in a single case.Conclusions: Sequencing HCMV genomes from cCMV FFPE material is feasible. This potentially facilitates larger studies of the effects of HCMV variation on the clinical outcome of cCMV.