Vipie: web pipeline for parallel characterization of viral populations from multiple NGS samples

Abstract

Background: Next generation sequencing (NGS) technology allows laboratories to investigate virome composition in clinical and environmental samples in a culture-independent way. There is a need for bioinformatic tools capable of parallel processing of virome sequencing data by exactly identical methods: this is especially important in studies of multifactorial diseases, or in parallel comparison of laboratory protocols.

Results: We have developed a web-based application allowing direct upload of sequences from multiple virome samples using custom parameters. The samples are then processed in parallel using an identical protocol, and can be easily reanalyzed. The pipeline performs de-novo assembly, taxonomic classification of viruses as well as sample analyses based on user-defined grouping categories. Tables of virus abundance are produced from cross-validation by remapping the sequencing reads to a union of all observed reference viruses. In addition, read sets and reports are created after processing unmapped reads against known human and bacterial ribosome references. Secured interactive results are dynamically plotted with population and diversity charts, clustered heatmaps and a sortable and searchable abundance table.

Conclusions: The Vipie web application is a unique tool for multi-sample metagenomic analysis of viral data, producing searchable hits tables, interactive population maps, alpha diversity measures and clustered heatmaps that are grouped in applicable custom sample categories. Known references such as human genome and bacterial ribosomal genes are optionally removed from unmapped ('dark matter') reads. Secured results are accessible and shareable on modern browsers. Vipie is a freely available web-based tool whose code is open source.

Keywords: Assembly; Metagenomics; NGS analysis; Parallel processing; Viral dark matter; Viromes; Virus; Visualization.

Fig. 1

Vipie web flow chart. For efficiency, sample based paired FASTQ files are uploaded as a zipped archive with optional mapping file. Illumina BaseSpace archive downloads can be used without changes. All pipeline parameters can be entered using the web form. The default values and use case are listed in the user guide available at home page along with example multi-sample archive input

Fig. 2

Interactive population profile maps and diversity. Vipie results are securely accessed and browser based. a Population chart slices are clickable and their sizes represent relative percentage of relevant taxonomy level. Diarrheal sample is dominated by dsRNA (orange) Rotavirus while African stool samples contain ssRNA (green) and dsDNA viruses. b Alpha diversity is calculated using Shannon entropy. Vipie charts are interactive and can be saved as multiple image formats

Fig. 3

Clustered heatmap of HMP, African and Japanese diarrheal samples. Public NGS data from different consortiums provide opportunities for advanced comparative virome analysis. Healthy HMP sample types clustered correctly (nasal, vaginal, blood samples) while a Japanese sample (gastroenteritis dataset from the VirusTAP report) and African samples (known to be positive for multiple viruses) showed different signatures. HMP samples can be identified using the legend on upper right, with olive green for nasal, yellow for vagina and blue for blood. Samples from rural Africa and VirusTAP (Japan) are marked in colors brick and red

Fig. 4

QC and distribution of reads including dark viral matter. a The chart shows the number of NGS reads retained per sample through QC, interlacing and de novo assembly. b Sample reads, along the x-axis and their aligned origins are shown as stacked bars. Shown in black, unmapped viral ‘dark matter’ is of high interest across virology studies. Blue bars represent bacterial ribosome, green for human while red is for known viral matches