Percentage of mapped reads to the HCMV transcriptome in primary cell types.

<p>Six different blood donors (A-F) were processed to obtain monocytes which were differentiated to DCs, MΦ1 and MΦ2. Subsequently, these primary cell types were infected with TB40/E at MOI5. As a comparison, we infected NHDF cells (n = 3) at MOI 0.16, MOI 0.5 and MOI 5. Given in the top panel are the percentages of mapped reads on the HCMV genome for each cell type.</p

HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types

<div><p>Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34⁺ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread <i>in vivo</i>. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential of HCMV to adapt to or influence different cellular environments to promote its own survival.</p></div

Expression levels of the HCMV genes transcriptome in fibroblasts (MOI 0.16).

<p>Log(10) of the RPKM values per gene were binned in six categories and mapped on the reference genome of TB40/E.</p

Enrichment of functional groups when comparing DCs with both types of macrophages and when comparing MΦ1 with MΦ2 macrophages.

<p>The HCMV was annotated and a mathematical model was used to determine which functional groups were significantly enriched in the differentially regulated genes. Based on this algorithm, genes involved in cell tropism, viral spread and immunomodulation are the most enriched groups in DCs, MΦ2 and MΦ1.</p

Enrichment of functional groups in DCs, MΦ1 and MΦ2 compared to NHDF.

<p>Genes involved in cell tropism, viral spread and immunomodulation are shown to be the most enriched groups in DCs, MΦ2 and MΦ1. Given are the p-vales of each individual gene.</p

Differential regulation of HCMV gene expression in monocyte-derived DCs, MΦ1 and MΦ2 (MOI5) compared to lytic infection in NHDF fibroblasts (MOI0.16).

<p>On the graph is the fold increase or decrease (reflecting genes which are expressed higher/lower in fibroblasts than in primary cell types). Red bars represent significant changes in gene expression between the indicated cell type and NHDF fibroblasts (adjusted p<0.05), black bars indicate modulated genes which did not reach the significance threshold (adjusted p≥0.05).</p

Additional file 5: of QQ-SNV: single nucleotide variant detection at low frequency by comparing the quality quantiles

Supporting evidence for applicability of QQ-SNV for SNV calling on Illumina NGS sequencing data of any read length. (PDF 32 kb

Assembly performance using 454 GS FLX, IGA or both and freeware or commercial software suites.

<p>Boxplots representing [A] the range of n50 contig lengths and [B] number of gaps in contig coverage of consensus sequences after <i>de novo</i> assembly of respectively 454 GS FLX, IGA or combined datasets. The central line in the box represents the median, top and bottom represent the 75 and 25 percentile and error bars represent minimum and maximum values. Median values are stated above each boxplot. Datasets (454 GS FLX and/or IGA) and software suites (<i>CLC Genomics Workbench</i>, <i>MIRA</i>, <i>Velvet</i> or <i>Phrap</i> combining <i>MIRA</i> and <i>Velvet</i> assemblies) are indicated below the plots. Since normality was violated, overall differences for n50 contig length and number of gaps were tested with the non-parametric Friedman test (n = 13; n50 contig length: χ²(5) = 42.506, p<0.001; gaps: χ²(5) = 37.275, p<0.001). Comparisons between assemblies based on different datasets were made using the Wilcoxon Signed Ranks Test with Bonferroni correction; p-values are reported in the figure. Because of the Bonferroni correction, differences are only significant when p<0.017.</p

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

<div><p>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 <i>in vitro</i> 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.</p></div

Comparison of strain BE/21/2010 consensus sequences, derived directly from the clinical material (BE/21/2010 up) and after four cell culture passages (BE/21/2010 p4).

<p>Comparison of strain BE/21/2010 consensus sequences, derived directly from the clinical material (BE/21/2010 up) and after four cell culture passages (BE/21/2010 p4).</p