A Method Enabling High-Throughput Sequencing of Human Cytomegalovirus Complete Genomes from Clinical Isolates

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous virus that can cause serious sequelae in immunocompromised patients and in the developing fetus. The coding capacity of the 235 kbp genome is still incompletely understood, and there is a pressing need to characterize genomic contents in clinical isolates. In this study, a procedure for the high-throughput generation of full genome consensus sequences from clinical HCMV isolates is presented. This method relies on low number passaging of clinical isolates on human fibroblasts, followed by digestion of cellular DNA and purification of viral DNA. After multiple displacement amplification, highly pure viral DNA is generated. These extracts are suitable for high-throughput next-generation sequencing and assembly of consensus sequences. Throughout a series of validation experiments, we showed that the workflow reproducibly generated consensus sequences representative for the virus population present in the original clinical material. Additionally, the performance of 454 GS FLX and/or Illumina Genome Analyzer datasets in consensus sequence deduction was evaluated. Based on assembly performance data, the Illumina Genome Analyzer was the platform of choice in the presented workflow. Analysis of the consensus sequences derived in this study confirmed the presence of gene-disrupting mutations in clinical HCMV isolates independent from in vitro passaging. These mutations were identified in genes RL5A, UL1, UL9, UL111A and UL150. In conclusion, the presented workflow provides opportunities for high-throughput characterization of complete HCMV genomes that could deliver new insights into HCMV coding capacity and genetic determinants of viral tropism and pathogenicity.

High-Throughput Analysis of Human Cytomegalovirus Genome Diversity Highlights the Widespread Occurrence of Gene-Disrupting Mutations and Pervasive Recombination

“…Samples were collected at the University Hospitals Leuven (n ϭ 81) and Saint-Pierre University Hospital (n ϭ 13) in Belgium and at the Institute of Hematology and Blood Transfusion in the Czech Republic (n ϭ 6). Virus culture, DNA purification, and amplification were executed as described previously (36). Briefly, samples were inoculated onto E 1 SM fibroblasts and cultured for the number of passages listed in Table S1 in the supplemental material.…”

“…Library preparation for 454 and Illumina sequencing was performed as described previously (36). Libraries were sequenced on the 454 GS FLX (Roche) and GAIIx and HiSeq2000 (Illumina) platforms (see Table S1 in the supplemental material).…”

“…Libraries were sequenced on the 454 GS FLX (Roche) and GAIIx and HiSeq2000 (Illumina) platforms (see Table S1 in the supplemental material). Full-genome consensus sequences were derived by using an approach that has been discussed extensively, with some modifications (36). This approach consisted of de novo assembly, scaffolding of contigs on HCMV reference sequences, and construction of a hybrid reference combining contig and background reference sequences.…”

“…Assemblies were then cut at these regions, and the separate contigs were extended and eventually joined by iterative mapping of sequence reads. Remaining uncertainties were resolved via PCR amplification and Sanger sequencing, as described previously (36). Data concerning the number of sequence reads mapping to the final genome consensus and average read depth are summarized in Table S1 in the supplemental material.…”

The Cell Biology of Cytomegalovirus: Implications for Transplantation

“…The application of genomic and transcriptomic highthroughput sequencing technologies in clinical infections are likely to elucidate mechanisms of viral pathogenesis and antiviral therapy (97)(98)(99). There is no clear correlation, for example, of specific genotypes for UL55 (gB), UL73 (gN), UL144 (TNF-a receptor) or UL75 (gH) with clinical manifestations of disease (38,100).…”