The DifferentialNet Database Tutorial

To go back to the DifferentialNet home page click here.

This tutorial offers an overviewof DifferentialNet, explanation of the iconswhich are constant across DifferentialNet pages, explanation about the different search optionsand the DifferentialNet output page.

Please see our FAQ page.

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Contact:  Esti Yeger-Lotem at estiyl@bgu.ac.il

Table of contents:

General Input section Output section
Overview Query DifferentialNet output page
Icons RNA-Sequencing datasets menu Network view
PPI data Tissues menu Control Bar
  Protein name Tabs view
  Filter interactions (%) Properties tab
  Sample Protein Gene Ontology tab
  Random Protein Differential tab
    Graph Options tab
    Mouse right-click menu

 

DifferentialNet overview:

The DifferentialNet database (https://netbio.bgu.ac.il/diffnet)

Icons Appearing on DifferentialNet web-pages:

These icons are constant across all DifferentialNet pages:

 – to DifferentialNet homepage from every page.

 – open DifferentialNet homepage in a new tab.

 – to tutorial from every page.

PPIs data:

PPI data were gathered and from four major databases (BioGrid, DIP, MINT and IntAct) using the MyProteinNet webserver [1], currently resulting in 243,706 interactions between 17,283 proteins.

Note: Most of the available PPI data are oblivious to alternatively-spliced isoforms. Therefore, DifferentialNet associates each protein-coding gene with a single protein product. For simplicity we refer to the gene and its protein product interchangeably.

Querying DifferentialNet:

Show differential protein interactions for your protein: The user selects an RNA-Sequencing dataset, tissue, protein and percentile threshold. The output is a network view of the query protein and its PPIs in the selected tissue. Only interactions whose weight percentile is ≤ threshold will be shown.

RNA-Sequencing datasets menu:

DifferentialNet uses tissue expression profiles from three datasets:

  • The Genotype-Tissue Expression (GTEx) consortium [2]. 421 RNA-sequencing profiles from 42 tissues were included. These samples were taken from individuals with death reason 1 (traumatic injury), and not from treated patients.
  • The Human Protein Atlas (HPA) project[3]. 192 RNA-sequencing profiles from 27 tissues were gathered.

In both datasets, raw counts were normalized for each data source using the TMM method by the edgeR package [4], to obtain the same library size for every sample. Genes with less than 1- raw counts in all samples were removed before normalization. For each tissue the normalized count for a gene was set to its median normalized count across all corresponding tissue samples.

 

Tissues menu:

Once a dataset is selected, the user can select a specific tissue from this dataset to obtain its associated PPIs. The tissues drop down list is searchable for convenience.

The output contains the query proteins and its PPI partners whose interactions weight percentile was at least as high (in absolute value) as the selected percentile threshold in the selected tissues.

 

Protein name:

The user can search for a protein by HUGO Gene Symbol, Ensembl gene IDs or by Entrez gene IDs. After the user has entered the gene name into the text box DifferentialNet will query its server to check if the gene is in the database. If the gene is not in the DifferentialNet database, the message “Your search returned no results” will be displayed. If the gene was found in the DifferentialNet database, it should be selected from the displayed list.

 

Filter interactions (%):

DifferentialNet offers the user the option to set the percentile threshold dynamically. Only interactions whose weight percentile (in absolute value) is ≤ threshold will be presented in the network. Interactions with weight percentile (in absolute value) higher than the threshold will be removed from the network along with their proteins.

Sample Protein:

This link displays the DMD protein in GTEx dataset with “Muscle – Skeletal” selected at a filtering percentile of 10. 

Random Protein:

This link displays a randomly selected protein. The RNA-Sequencing dataset, tissue and threshold are all set randomly.

DifferentialNet Output Page:

DifferentialNet output page offers several parts: The Network View, the Tabs Viewand the Mouse right-click menu. Here we offer explanations to these parts.

Network View:

The network view is an interactive graphical representation of the output, displayed using Cytoscape.jsplugin [5]. Nodes represent proteins, and edges between them represent their experimentally detected tissue-associated PPIs.

In this view, proteins are shaped to highlight disease related proteins from OMIM, and interactions are colored to indicate their differential weight in the tissue. This view enables users to quickly assess the differentiality or global functionality of the output network.

Figure 1. View legend

Control Bar:

i: The information button

 

Tabs View:

Users can retrieve various attributes of selected proteins and interactions through the tabs view on the right. The available tabs are Properties, Gene Ontology, Tissue Expressionand Graph Options.

Properties tab:

This tab shows information about selected proteins and interactions.

Proteins have the following attributes:

  1. HUGO Gene symbolof the protein.
  2. Ensembl Gene IDof the protein.
  3. Entrez IDof the protein.
  4. MIM Accessionnumber, if the protein is associated to a disease, this data is obtained from the OMIM database. If the protein is not associated with a disease N/A will be displayed.
  5. MIM Description if the protein is associated to a disease, this data is obtained from the OMIM database. If the protein is not associated with a disease N/A will be displayed.

Interactions have the following information:

  1. Protein 1: HUGO gene symbol of one of the interacting proteins.
  2. Protein 2: HUGOgenesymbol of the second interacting protein.
  3. Differential Weightof the interaction in the selected tissue.
  4. Percentile of the interaction weight in the selected tissue interactome.
  5. Tissue Differentiality:numberof tissues in which the interaction weight is below the selected threshold.
  6. Detection Method(s):Provides the experimental method(s) by which the interaction was detected (e.g., Affinity Capture-Western); whether this was a low-througthput (Low) or high-throughput (High) experiment; and the database reporting this PPI (e.g. BioGrid).

Gene Ontology tab:

This tab shows Gene Ontology (GO) annotations for selected proteins. Each GO annotation appears with its GO id, description and evidence code. GO annotations are retrieved from AMIGO using the MyGene.infoweb-service [6].

Differential tab:

This tab provides the weight and percentile values for selected interactions in all the different tissues. Tissues in which the interactions percentile is above the threshold are highlighted in red.

Graph Options tab:

This tab offers several ways to manipulate the network view.

Change Network Layout:allows the user to select a different network layout. The supported layouts are: (i) spread, in which the proteins are spread evenly around the query protein, (ii)  random, in which proteins appear in random order in the network view, (iii) grid, in which proteins are ordered evenly on a grid, and (iv) circle, in which proteins are ordered on a circle.

Network Options: Includes three features
(i) Remove Selected Proteins – remove the selected proteins and their interactions from the network
(ii) Select an additional query protein – upon selection of a network protein, adds its tissue-associated PPIs to the current network. This option is available for a single protein at a time,
(iii) Export to GraphML – download a GraphML file for the network, to be used with other network applications, such as Cytoscape.

Mouse right-click menu:

Clicking the right mouse button opens a menu with the following options:

When clicking on a node:

  1. Select as an additional query protein: Upon selecting a protein in the network, this option allows you to add its tissue-associated PPIs to the current network.(this option allows the addition of a single protein each time).
  2. Remove Selected Proteins removes your selected proteins and their PPIs from the network.

When clicking anywhere else:

  1. Export to GraphML lets you export the information of the proteins and PPIs to GraphML format. This format can be loaded into Cytoscape.
  2. Layoutslets you choose a different layout for the graph. By default, spread layout is selected.

REFERENCES:

  1. Basha, O., et al., MyProteinNet: build up-to-date protein interaction networks for organisms, tissues and user-defined contexts.Nucleic Acids Res, 2015. 43(W1): p. W258-63.
  2. Consortium, G., The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans.Science, 2015. 348(6235): p. 648-60.
  3. Uhlen, M., et al., Proteomics. Tissue-based map of the human proteome.Science, 2015. 347(6220): p. 1260419.
  4. Robinson, M.D., D.J. McCarthy, and G.K. Smyth, edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics, 2010. 26(1): p. 139-40.
  5. Franz, M., et al., Cytoscape.js: a graph theory library for visualisation and analysis.Bioinformatics, 2016. 32(2): p. 309-11.
  6. Wu, C., I. MacLeod, and A.I. Su, BioGPS and MyGene. info: organizing online, gene-centric information.Nucleic acids research, 2013. 41(D1): p. D561-D565.
  7. Kuhn, M., et al., STITCH 3: zooming in on protein-chemical interactions.Nucleic Acids Res, 2012. 40(Database issue): p. D876-80.