Overview

Two types of viral proteomic tree: "reference tree" and "user tree"

As a preset of the ViPTree server, reference viral genomes stored in Virus-Host DB (see Dataset used in ViPTree for details) are classified into six categories mainly based on their nucleic acid types: (1) dsDNA, (2) ssDNA, (3) dsRNA, (4) ssRNA, (5) ssRNA-RT, and (6) dsRNA-RT. Viral proteomic trees for each category (called as "reference tree") are pre-calculated from all-against-all genomic similarity scores (S_G) computed by results of tBLASTx. In addition, viral proteomic trees of host-specific subsets (i.e., eukaryotic/prokaryotic subsets) of each category are also available. These can be browsed in the proteomic tree viewer from the "reference trees" dropdown menu in a navigation bar on the top. For details about the proteomic tree viewer, see the section of proteomic tree view. Method for computation of S_G is described in Bhunchoth et al., 2016.

Users can generate their viral proteomic trees (called as "user trees") by uploading genome sequences and selecting one of the reference tree from the upload page by choosing "With references" mode. The resulting user tree includes user's viral genomes and reference virus genomes included in the selected reference tree. By choosing "Only query" mode, users can generate a user tree that includes only user's viral genomes. After generation of a user tree, a subset of viruses is automatically selected by genomic similarity to the user's viruses and can be manually selected for regenerating a smaller "focused" proteomic tree for further investigation and publication-ready visualization. For details about user trees, see the section of user tree generation.

Genomic alignment view

From alignment viewer, users can browse an alignment of any set of viral genomes included in a reference tree or a user tree (see example: alignment of three reference viral genomes). Viral genomes included in the alignment can be flexibly selected (e.g., by clicking an inner node of a proteomic tree or using check box in a genomic table). In the alignment, high-similarity regions detected by tBLASTx are color-coded reflecting on the reported %-identity. In addition, dot plots summarizing these high-similarity regions are shown beside the alignment. Automatic position adjustment on the alignment (including adjustment of circular permutation, which is frequently observed in viral genomes) are implemented for intuitive visualization (e.g., for quick understanding of genomic collinearity). Manual position adjustment can also be applied. For details of genomic alignment view, see this section.

Gene prediction and similarity search

After upload of viral genomes, gene prediction on the genomes and protein similarity search against GenomeNet nr-aa, a non-redundant protein sequence database merging sequences of RefSeq, SwissProt, TrEMBL, and GenPept, are also performed in parallel with proteomic tree construction. The result can be browsed through the web interface and downloaded as tables. In the genomic alignment view, resulting gene positions in uploaded genomes and best hits against nr-aa are used to indicate gene positions and labels. For the reference viral genomes, gene positions and annotations are retrieved from NCBI flat files.

Dataset used in ViPTree

Viral genome sequences and taxonomic information of viruses and their hosts are based on the Virus-Host DB. Virus-Host DB covers viruses with complete genomes stored in (1) NCBI RefSeq and (2) GenBank whose accession numbers are listed in EBI Genomes. The host information is collected from RefSeq, GenBank (in free text format), UniProt, ViralZone, and manually curated with additional information obtained by literature surveys. Following Virus-Host DB update in every couple of months, viral genome information used in ViPTree will be updated.

For preparation of all-against-all genomic similarity calculation, segmented viral genome sequences are concatenated into one by inserting 100 ambiguous nucleotides (N) at each concatenation site for the purpose of proteomic tree calculation and genomic alignment visualization. These types of sequences are indicated in the ID field as "comb-XXXX" (e.g., comb-NC_007548).

Resource for computation

The ViPTree server is a part of GenomeNet service. The computational time is provided by Supercomputer System of the Institute for Chemical Research, Kyoto University.

Proteomic tree

The page of the proteomic tree view contains (1) the viral proteomic tree, (2) a virus genome table, and (3) a panel for configuration / download.

Viral proteomic tree

Viral proteomic tree is shown on the right of the screen (or on the bottom when the screen width is short). A user can select one from two types of tree view: "circular view" and "rectangular view". The circular view is designed for a comprehensive visualization of a proteomic tree regardless of the number of viruses (even in the case of thousands of viruses). On the other hand, the rectangular (i.e., linear) view is suitable for browsing detailed information. When the number of viruses are small enough, the rectangular view could be comprehensive. In both types of views, information of virus families and host taxonomic groups (for the most case at phylum-level, except for Proteobacteria at class-level) are represented by color rings or lines.

An example of the circular view is shown here. The tree is a reference tree of prokaryotic ssDNA viruses.
Here is the same tree visualized in the rectangular view.

In the rectangular view, each of inner nodes represented by filled circles is linked to an alignment of genomes that are included in its subtree. Such inner node hyperlink can be shown in the circular view by a parameter "show link to alignment" inside the configuration panel. This panel provides various functions for configuration of a proteomic tree as well as image/data download. For details of the panel, see this section.

Note about the appearance of proteomic trees

Since ViPTree generates a rooted proteomic tree using "midpoint rooting", the location of the root is operationally determined. It may not reflect the directonality of evolution within the tree, especially when "Only query" mode is selected.

Virus genome table

A genome table, shown on the left (or on the top when the screen width is short) of the proteomic tree, consists of six columns as follows:

check box, genome id, taxonomy id, virus name, virus family, host group.

A genome table is initially sorted by the order of genomes appeared in the proteomic tree. By clicking each header of the table's columns, the genomes can be sorted in an ascending/descending order of the column. The number of lines shown in a page can be changed by the selector on the top left of the table. A text box for free text search is available on the top right of the table. For the columns of virus families and host groups, a filtering can be performed by selectors at the bottom of the table.

By clicking an "add stars" button above the table, selected genomes by check boxes will be highlighted with red stars in the tree. On the other hand, by clicking a "browse an alignment" button, a genomic alignment of viruses selected by check boxes will be generated.

An example using the genome selection function is shown here. This is a reference proteomic tree of prokaryotic ssDNA viruses with six Chlamydia phages highlighted. In this page, by clicking a "browse an alignment" button, a genomic alignment of these six Chlamydia viruses can be browsed. For details and features of the genomic alignment view, see this section.

Panel for configuration / download

A panel for configuration / download is also shown on the left (or on the top when the screen width is short) of the proteomic tree. This panel provides a switch between the circular view and the rectangular view by using "Circular tree" tab and "Rectangular tree" tab on the top of the panel and by clicking the "redraw tree" button. "Download" tab provides download links for the genome table, visualization and tree files.

This panel also provides many visualization parameters for a proteomic tree.

"Circular tree" tab and "Rectangular tree" tab provide functions listed below.

branch length scaling (log/linear/non-scaled)—Representation of branch length scaling can be changed.
genome table (shown/not shown for speedup)—A genome table can be hidden for faster redraw of the proteomic tree.
show link to alignment (shown/not shown)—Links to a genomic alignment are shown or hidden.
show labels of genomes (full description/virus name/not shown)—Labels of each genome can be shown (full description/virus name) and hidden.
tree radius and space for labels—Size of tree drawing and space for label descriptions.
base font size, font family, and font weight—Parameters for font settings.

"Download" tab provides three download links listed below.

A table of genomes in the tree, including various information. In case that the tree includes uploaded sequences, genomic similarity scores (S_G) of the genomes to any genomes included are also listed.
A Newick formatted file of the proteomic tree (calculated by BIONJ based on genomic distance matrix)
An image file of current tree view (SVG format)

After the download of the SVG file, SVG formatted images can be edited and/or converted to other formats (e.g., PDF, PNG and TIFF), by software such as Adobe Illustrator and Inkscape, which is freely available for Windows, macOS, and Linux PC.

Genomic alignment

The page of the alignment view contains (1) panel for configuration/download and (2) the alignment view.

In the alignment view, a user can browse an alignment including reference viral genomes, as well as genomes uploaded by the user. The alignment view visualizes homologous regions between genomes detected by tBLASTx (E-value < 1e-2). An example of the genomic alignment view is shown here.

Caution: Microsoft Internet Explorer may take a long time to visualize genomic alignments. Please use other browsers, such as Google Chrome (recommented), Edge, Firefox, Safari, etc.

This view also provides pairwise dot plots of sequences included in the alignment. A color bar on the upper left of the alignment represents %-identity shown in the alignment and dot plots. This bar can be enlarged/shrinked by scrolling mouse wheel on the bar.

Panel for configuration/download

A panel for configuration/download is shown above of the alignment image. The panel contains three navigation tabs: "Basic parameters", "Customize sequences", and "Download". These tabs provide versatile functions for publication-ready visualization of the alignment.

"Basic parameters" tab provides parameters related to genome positioning, gene labels, size adjustment, etc.

- positioning of sequence (auto/as is/reset)—switch for auto adjustment of alignment position for clear representation of sequence collinearity (i.e., circular permutation, reverse orientation, and shift of the start position)
- gene labels (shown/not shown)—switch for gene labels shown over arrows representing genes
- gene labels angle—Gene labels can be rotated.
- gene pop-up (shown/not shown)—switch for pop-up listing gene information
- BLAST %-identity interval (default/customize)—%-identity corresponding to the alignment colors can be customized by inputting comma-separated multiple values (0 to 100; decimal point is accepted).
- BLAST pop-up (shown/not shown)—switch for pop-up listing BLAST information
- BLAST %-identity colors (default/rainbow/iridescent/monochromed/customize)—the figure can be monochromed or alignment colors can be customized by inputting one color (used as seed) or comma-separated multiple colors (all colors specified). If one color is specified, gradation from the given color to white is generated. When more than 10 intervals are given in "%-identity interval" section, "rainbow colors" will be used as default.
- BLAST visualization (normal/position emphasized)—"Position emphasized" is purposed to check exact positions of BLAST hits.
- ticks of genomes / vertical dashed lines (shown/not shown)—ticks and vertical dashed lines interval for each sequence
- BLAST %identity / score / length threshold—thresholds for visualization of BLAST result
- scale of sequences—parameter for enlargement/shrinkage of displayed sequences
- interval between sequences—vertical space length between sequences
- scale of vertical dashed lines—scale for vertical dashed lines
- interval of dotplot grid—grid interval in dot plots
- font family, base font size and font weight—parameters for the font setting

"Customize sequences" tab provides a table to add/alternate/delete sequences that are included in the alignment and reorder of sequences. Each genome in the alignment can be manually/automatically repositioned by circular permutation, reverse stranded and shift of start position.

"Download" tab provides a download link of a represented alignment image in the SVG format.

User tree generation

Prepare sequences

From the upload page, users can upload viral genome sequences and choose nucleic acid types and host categories (i.e., prokaryotes/eukaryotes; optional), to generate a proteomic tree together with reference viral genomes. In "Only query" mode, users can generate a user tree that includes only user's viral genomes. After validation of the uploaded sequences, a new session will be created and computation begins.

Current limitation for the number of sequences and ID constraints are listed below.

Input FASTA should include genomic nucleotide sequences (up to 300 sequences).
When "Only query" mode is chosen, 3 or more sequences should be uploaded.
Each sequence should be no longer than 20 million nucleotides and no shorter than 100 nucleotides.
Sequence IDs are recommended to be within 30 characters. If longer than that, the first 30 characters will be used.
Alphabets, digits, and special characters (dots, hyphens, and underscores) can be used as sequence ID. The other characters will be replaced with underscores.
If sequence ID begins or ends with the special characters, the special characters will be removed.
If you want to upload and compare multi-contig genomes, try ReCCO (beta version) to rearrange and concatenate the contigs.

Gene information

There is options for gene finding: (1) use Prodigal to predict genes, (2) upload pre-defined BED-like formatted gene position table, and (3) without gene information.

If prodigal is used for gene prediction, coding table can be selected. An important note is that .....

When users would like to use their predefined gene information, a gene table can be uploaded. The table should be tab-separated, expanded BED-like format composed of columns as follows.

genome sequence ID
starting position of gene (0-based position)
stopping position of gene
gene ID (acceptable characters: digits, alphabets, dots, hyphens, and underscores)
anything (not used by ViPTree)
strand (+/-)
(optional) description label used in alignment viewer. If this is not given, GHOSTX result is used (default behavior).
(optional) color name/code used in alignment viewer. If this is not given, default color is assigned.

Be careful for the 2nd column. If a gene starts from the first nucleotide, the value should be 0 (not 1), while the 3rd column does not have to be changed: if a gene stops at the 90th nucleotide, the value should be 90.

Gene function prediction

ViPTree can perform similarity search against GenomeNet nr-aa, a non-redundant protein sequence database merging sequences of RefSeq, SwissProt, TrEMBL, and GenPept, using GHOSTX (evalue cutoff: 0.1).

It will take a relatively long time to other computation performed by ViPTree. Users can skip this process.

If the similarity search was performed, users can browse the resulting table (Example).

The top hits can be browsed, up to 100 hits, if exist. The GenomeNet nr-aa database is currently weekly updated by GenomeNet. The details of hit sequences can be browsed using a hyperlink.

Computation steps

Computation steps include tBLASTx execution, distance matrix generation, proteomic tree generation, gene finding, and gene similarity search. Clicking the submit button on the upload page makes redirection to a calculation progress reporting page like this. The page will be automatically reloaded to announce the progress under calculation. When all the calculation steps on the upload are finished, a "session" is activated. At that time, a notification email is sent to the uploaded address. Users are able to start browsing all the results from the session main page that is announced in the progress page and the email. An example of the session main page is shown (Example).

Computation time will depend on various factors. Generally, the sum of length of uploaded sequences is one factor. This is because the sequence length affect on computation time of the tBLASTx and the number of genes affects on time of gene similarity search by GHOSTX against GenomeNet nr-aa. We tested various input sequences, and in most cases, calculations were finished from minutes to a couple of hours (except when the computer system is fully occupied).

Browsing session

The session main page is a portal for investigation of the results (Example). On the left of the screen (or on the top when the screen width is short), basic information of the session is listed. On the right of the screen (or on the top when the screen width is short), menu for all the viewer and file download are provided.

Contents of the basic information

Uploaded sequences and the selected dataset of reference genomes
Selected options about gene prediction
Time of data uploaded and calculation finished.
Session ID and ViPTree release by which the session is generated

Note about update recommendation

If ViPTree is updated after the session creation, session might not work properly. The new version may include a new feature that is not compatible with the session, which is built in the older version. Also, data compatibility of the session might not be reliable since genome information could be changed from the update.

Contents of the menu

Browse proteomic tree (related genomes / all genomes)
Regenerate proteomic tree (make subset)
Browse regenerated (subset) tree
Browse table (genomic similarity to reference genomes and gene similarity search)
Download files (zip file containing blast outputs, genomic similarity, gene prediction, and gene similarity search, or all of them)

Proteomic tree of related genomes

It could be ineffective to browse the proteomic tree including all the genomes when a user is only interested in genomes close to the user's ones, which is considered to be a typical case, and when generated tree contains thousands of sequences (e.g., when the user selects a reference set of dsDNA prokaryotic viruses). For such a case, the proteomic tree of "related genomes" enables effective investigation. The tree contains user genomes and related reference genomes that are related to the user's ones in terms of genomic similarity. The related reference genomes consist of two categories as follows.

reference genomes that show sequence similarities (S_G > 0.02) to at least one of the user genome
reference genomes that show sequence similarities (S_G > 0.02) to at least one of the reference genome of category 1.

Tree regeneration (extraction of a subset)

Users can make a selection of genomes that are a subset of the produced tree proteomic tree) including only interested genomes ("regenerated tree"). This function provides the way of focused investigation on the interested viruses and generation of publication-ready figures. This selection can be done from "Regenerate proteomic tree" links in menu. Two options are provided for selection of genomes: (1) by selecting genomes using check box in the genome table or (2) by pasting ID list. Tree regeneration generally takes a few minutes for the computation, and a notification email will be sent when the computation finished. This operation can be repeated, the resulting trees can be browsed and these visualization can be downloaded.

Licensing

All data and download files in ViPTree are freely available under a 'Creative Commons BY-NC-SA 4.0' license.

When using the data and images, please provide appropriate credit, provide a link to the license, and indicate if changes were made.
Do not use the material for commercial purposes.
If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original.