NetCirChro Manual







User Manual

Version 1.2




Bioinformatics and Computational Biosciences Branch (BCBB)
OCICB/OSMO/OD/NIAID/NIH

April 2012

Introduction to NetCirChro

The abundance of complete, annotated microbial genomes has enabled the reconstruction of biological networks that incorporate diverse datasets including annotation, expression, and biochemical and physiological characterizations. Analysis of these networks assists in the identification of gene targets and the generation of new hypotheses about their functions. Since the functional association between genes may be inferred from chromosomal location and proximity, an interactive tool for visualizing and analyzing network data in the spatial context of the chromosome was needed. The NIAID Bioinformatics and Computational Bioscience Branch (BCBB) has developed the Networks on Circular Chromosome (NetCirChro) tool to enable users of the network visualization platform Cytoscape to overlay networks onto a circular chromosomal map. The synchronization of data selection between NetCirChro and Cytoscape allows users to leverage the rich Cytoscape features to navigate, search, filter, and highlight complex datasets. The unique NetCirChro analysis features support identifying user-specified gene coordinate patterns and network topology patterns on the chromosome. NetCirChro will help scientists discover the potential role of gene organization in functional regulatory networks. It will also assist in understanding the functional properties of neighboring genes.

Installation

Quick Start

NetCirChro supports the Cytoscape session management system. To download the NetCirChro plugin and an example session file, please visit NetCirChro official site http://exon.niaid.nih.gov.

After you copy NetCirChro plugin into the Cytoscape plugin folder, you can use Cytoscape menu "File | Open" to open the session file.

Launch NetCirChro

NetCirChro can be launched from Cytoscape menu "Plugin | NetCirChro".


Figure 1 NetCirChro plugin
Figure 1 NetCirChro plugin


The genome data panel will be displayed in the Cytoscape control panel.


Figure 2 Getting genome data
Figure 2 Getting genome data


NetCirChro can download the data from NCBI and read data from the local files. NetCirChro can also get the data from Cytoscape sessions. Currently, NetCirChro supports GenBank format file (.GBK) or protein table format file (.PTT).

Downloading Genome Sequence

NetCirChro downloads data files of complete microbial genomes from http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi?view=1.

  1. Search

    After the button "Import from NCBI..." in the Cytoscape control panel is clicked, NetCirChro will display a download window for microbial genomes. The window displays a list of microbial genome projects. You can search any field in the window.


    Figure 3 Searching complete microbial genome data
    Figure 3 Searching complete microbial genome data


  2. Download

    Once you select an organism, you can click "Download" button to retrieve the data. In the popup window, the available chromosomes of the organism will be displayed. You can choose one or more chromosomes to download.


    Figure 4 Downloading selected microbial data
    Figure 4 Downloading selected microbial data


  3. Display

    Once the data is downloaded, NetCirChro will automatically read the file and visualize the chromosome.


    Figure 5 Displaying the data
    Figure 5 Displaying the data


Displaying and Manipulating Diagrams

You can use NetCirChro to control the visual appearance of the chromosome diagrams and networks.

NetCirChro Window

NetCirChro Menu

Mapping Network

Controlling Visual Appearance

Selecting Network

Selecting Data

Adding Another Chromosome

Creating Subnetwork

Saving and Opening Session

Zooming Diagram

Saving and Printing Image

NetCirChro Window

The main window of NetCirChro has similar structure as Cytoscape window. The left side of the main window is the control panel and bird's eye view. The control panel is used to adjust the diagram appearance. Birds' eye view helps you to navigate different part of the diagram if it is zoomed to a large size.

At the right side are the visualization panel and data panel. The chromosome and network are displayed in the visualization panel. Data panel displays the GenBank annotation of the selected data in the visualization panel.


Figure 6 NetCirChro main window
Figure 6 NetCirChro main window


NetCirChro Menu

Here is a quick tour of the NetCirChro menus.

File Menu

Edit Menu

View Menu

Search Menu

Statistics Menu

Window Menu

Help Menu

Mapping Network

ID Mapping

After the chromosome data is loaded into NetCirChro, you need to decide how the nodes in the Cytoscape networks are mapped to the chromosome features in NetCirChro. Figure 7 shows the qualifiers of features in a chromosome. Figure 8 shows the node attributes of a network. In this example, the content of the qualifier "locus tag" is same as the content of the node attribute "canonicalName". Therefore, you need to specify these two fields in the mapping dialog.

However, you may not get the complete mapping by just using one qualifier. For example, some nodes may use a gene synonym instead of locus tag as the canonical name. Therefore, you may have to specify multiple qualifiers to map the node attribute. NetCirChro enables you to use ordered qualifiers to map a node attribute. If the node attribute cannot be found in the first qualifier of all features, NetCirChro will try to map it to the second qualifier you specified, and so on.


Figure 7 Qualifiers in NetCirChro
Figure 7 Qualifiers in NetCirChro



Figure 8 Node attributes in Cytoscape
Figure 8 Node attributes in Cytoscape


Mapping Network Setting

You can get the mapping network dialog by clicking menu "Edit | Map Network..." In the dialog, you need to specify a network, a node attribute and one or more qualifiers for mapping.


Figure 9 Mapping network dialog
Figure 9 Mapping network dialog


Controlling Visual Appearance

NetCirChro enables you to control many visual appearance of the chromosome-network diagram from the track color, location, position to block arrow, label and color.

Terminology

The functional annotation of GenBank data file is organized as features. Each feature has a feature key to describe the specific annotation. Standardized qualifiers gives the detail description of the annotation. Since the qualifiers are standardized and the qualifier values usually are controlled vocabulary, the annotation can be easily parsed and processed. You can get the detail annotation organization from http://www.ncbi.nlm.nih.gov/projects/collab/FT/index.html.

Track Setting

A track is a ring-shape which represents selected feature sets of the chromosome. In the track setting window, you can add or delete a track. For each track, you can specify the width, position, and color of the current track. You can also specify which features are displayed in the track. For any changes in the track setting, you need click "Update" button to enable the changes on the diagram.

Track setting can be saved by clicking "Save" button. This setting will be the default setting for any following circular diagram.


Figure 10 Track setting
Figure 10 Track setting


Block Setting

A block is a small rectangle (or a line) which represents a feature of the chromosome. In the block setting window, you can get the name and location of the feature. The color, width, arrow and label display can be changed in this window.


Figure 11 Block setting
Figure 11 Block setting


Global Setting

In the global window, you can change several options for the whole circular diagram.


Figure 12 Global setting
Figure 12 Global setting


Selecting Network

When you select a tab in NetCirChro, the associated network in Cytoscape will be automatically selected and moved to front if necessary. On the other hand, if you select a network in Cytoscape and there is only one tab in NetCirChro which has the mapped network, the tab will be automatically selected.

When you close a tab in NetCirChro, if the network is not mapped to other chromosomes, NetCirChro will prompt a dialog to let you close the associated network in Cytoscape. If you try to close all tabs in NetCirChro, NetCirChro will also prompt a dialog to let you close all networks in Cytoscape.

Similar to Cytoscape, NetCirChro has a network list panel in the control panel. You can use this panel to select, rename and destroy networks.


Figure 13 Network list panel
Figure 13 Network list panel


Selecting Data

After the network is mapped to the chromosome, the selection in Cytoscape and NetCirChro is always synchronized. In both visualization panels, the selected nodes (blocks) and edges are highlighted. Meanwhile, the data panel displays the corresponding attribute values and annotations.


Figure 14 Selection synchronization
Figure 14 Selection synchronization


Since the data selection is synchronized between Cytoscape and NetCirChro, you can select, search or filter data from either Cytoscape or NetCirChro.

There are several ways to select data in NetCirChro.

  1. In NetCirChro, you can use mouse to drag a region on the diagram. The enclosed blocks and edges will be selected.


    Figure 15 Select data by mouse dragging
    Figure 15 Select data by mouse dragging


  2. Similar to Cytoscape, you can also choose data from data panel by right clicking and choosing "Select from table" menu.


    Figure 16 Selection from table
    Figure 16 Selection from table


  3. All search results in NetCirChro will be automatically selected and highlighted.


    Figure 17 Selection from searching
    Figure 17 Selection from searching


After the selection, the number of the selected nodes and edges are displayed in the network list panel of the control panel. You can also move the mouse outside of the circular diagram. A small yellow window will be displayed to show the mapping setting and the number of the selected nodes and edges.

Adding Another Chromosome

NetCirChro supports multiple chromosomes in one diagram. The additional chromosome, by default, will be displayed as a new track from the same starting position as the existing tracks. In addition, any newly-added chromosome should have smaller length than the first chromosome.

You can use menu "Edit | Add Chromosome..." to add a new chromosome. When the chromosome is added, the mapping between the network and the chromosomes will be updated automatically.

Creating Subnetwork

When a subset of data is selected in a network, you can create a subnetwork in Cytoscape. Similarly, NetCirChro provides a subnetwork creation feature. You can use menu "Edit | Create Network" to create a subnetwork from the selected blocks and edges in the current circular chromosome diagram.

There are several options for subnetwork creation.

Saving and Opening Session

NetCirChro supports the session saving and restoring of Cytoscape. The session file contains all the network data, chromosome data, visualization setting, and selection status.


Figure 18 Session menu
Figure 18 Session menu



Figure 19 Open session button
Figure 19 Open session button


Zooming Diagram

To zoom in the current diagram, you can use the shortcut key Command-I (Mac OS X) or Control-I (Windows). To zoom out the diagram, you can use Command-O (Mac OS X) or Control-O (Windows). Finally, if you want to display the whole diagram in the current tab, you can use the menu "View | Fit Window".

Saving and Printing Image

You can use the menu "File | Save Image As..." to export the image of the current diagram. NetCirChro supports many popular image formats including JPEG and PNG.

If you want to export the high-quality image for publication purpose, you can export the image as SVG or PDF format.


Figure 20 Save image menu
Figure 20 Save image menu


Link Out

NetCirChro provides "link out" feature similar to that of Cytoscape. When you select an gene in the NetCirChro data panel and right click it, a link out menu will be automatically displayed which allows you to search the gene in a variety of online databases.


Figure 21 Link out
Figure 21 Link out


Data Panel

In addition to the selection and link out feature, NetCirChro data panel allows you to display detail qualifier data and export the selected genes to a text file.

Display Qualifier Value

By default, NetCirChro only display the gene name and functional annotation in the data panel, which are corresponding to the locus tag and notes in the qualifiers. You can right click the data panel and choose "Customize columns" to display other qualifier values.


Figure 22 Customize columns menu
Figure 22 Customize columns menu



Figure 23 Column selection window
Figure 23 Column selection window


You can also check the detail of selected features by clicking the popup menu "Show detail". In the detail window, the GenBank annotation of selected features are displayed.


Figure 24 Qualifer detail window
Figure 24 Qualifer detail window


Export Data

The popup menu of the data panel contains a menu "Export data". This allows you to export the selected genes in NetCirChro to a tab delimited text files.

Searching Data in NetCirChro

NetCirChro provides several methods to search genes in the chromosome.

Searching Gene by Name and Function

Searching Gene by Coordinate

Searching Coordinate Pattern on Chromosome

Searching Paths in Network

Searching Neighborhoods in Network

Searching Gene by Name and Function

NetCirChro provides powerful Boolean search capability for gene names and functional annotations. NetCirChro recognizes the searching expression formed by AND, OR, NOT, and parentheses.


Figure 25 Search names or functions
Figure 25 Search names or functions


Searching String Example

Select Subset of the Results

After you click "Search" button to get the query result, you can choose one or more by clicking the checkbox in the "Select?" column. Then, you can click "OK" button to display the selected data in the NetCirChro main window.

Searching Gene by Coordinate

You can search and highlight genes based on their coordinates. The input can be a single number, or a range by connecting the start and end position with a hyphen. You can separate multiple ranges by using comma.


Figure 26 Coordinate range search window
Figure 26 Coordinate range search window


Searching Coordinate Pattern on Chromosome

Coordinate pattern search provides a powerful tool to search the spatial pattern in the network. It enables the users to specify the spatial pattern with the length of gene patterns and the gap between genes.


Figure 27 Coordinate pattern search window
Figure 27 Coordinate pattern search window


Terminology

Length and Degree are used to describe the contiguous node pattern with respect to position in the chromosome.

Example:

Backbone --------

Length=1 --++----

Length=2 --+++---

Length=4 --+++++-

Degree=1 ++------

Degree=2 +-+-----

Degree=3 +--+----

Search Options

In the search window, you can specify the length and degree of the pattern. Meanwhile, you can choose how the pattern is searched in the network.

Searching Paths in Network

NetCirChro can find a path or a loop in the network. First, you need select one or more genes in the diagram. NetCirChro will find all paths or loops starting from the selected genes.

In the path finder window, you can specify the length of the path or loop. A large length may take a long time to complete the searching. NetCirChro will prompt a waiting dialog which displays the current found path number. This waiting dialog also allows you to cancel the searching if necessary.


Figure 28 Path finder window
Figure 28 Path finder window



Figure 29 Waiting dialog
Figure 29 Waiting dialog


Searching Neighborhoods in Network

NetCirChro can find all neighbors nodes and adjacent edges of selected nodes in a network. This feature is a combination of Cytoscape menu "Select | Nodes | First neighbors of selected nodes " and " Select | Edges | Select adjacent edges"


Figure 30 Searching the neighbor nodes and edges
Figure 30 Searching the neighbor nodes and edges


Statistics in NetCirChro

NetCirChro provides several statistics for the chromosome and network data. Beyond the conventional statistics, NetCirChro can visualize the quantitative results on the diagram.

Gene Distance Distribution

Degree Distribution

Shortest Path Distribution

Comparative Distribution Chart

Cluster Coefficient and Coordinate Closeness

NetworkAnalyzer

Gene Distance Distribution

Gene distance means the coordinate distance between two genes of a chromosome. NetCirChro calculates the distance between every gene pair, if their products have an interaction in the protein-protein interaction network.


Figure 31 Gene distance distribution
Figure 31 Gene distance distribution


Based on the distance distribution, NetCirChro visualizes the distance on the circular diagram. If you click the button "Map to Circular Genome", NetCirChro will create a new map in the VizMapper. The value of gene distance is mapped to a continuous color range. The, the color of the edges in the Cytoscape and NetCirChro represents the distance between the two nodes/blocks. From the diagram, you can find that most interactions occur between gene products which are close to each other in the chromosome.


Figure 32 A new map of gene distance in VizMapper
Figure 32 A new map of gene distance in VizMapper



Figure 33 Mapping gene distance to edge color
Figure 33 Mapping gene distance to edge color


Degree Distribution

In a network, degree means the connection number of a node. NetCirChro calculates the degree of each node in a network and plots the node number of each degree in the chart.


Figure 34 Degree distribution
Figure 34 Degree distribution


If you click the button "Map to Circular Genome", NetCirChro will create a new map in the VizMapper. The value of degree is mapped to a continuous color range. Then, the color of nodes in Cytoscape and the color of blocks in NetCirChro represent the degree number. From the diagram, you can quickly identify the network hub in the chromosome.


Figure 35 A new map of degree distribution in VizMapper
Figure 35 A new map of degree distribution in VizMapper



Figure 36 Mapping degree distribution to node color
Figure 36 Mapping degree distribution to node color


Shortest Path Distribution

Any two genes (or their products) in a network have one or more paths. This box plot chart displays the shortest path length versus their distances on the genome. The box plot chart displays the statistical distribution of the gene distances on shortest path length. The bottom and top of the box shows the 25th and 75th percentile (the lower and upper quartiles, respectively), and the band near the middle of the box shows the 50th percentile (the median). The T shape ends show the minimum and maximum of the data.


Figure 37 Shortest path length distribution
Figure 37 Shortest path length distribution


Since it takes a long time to calculate all shortest path lengths in a large network, NetCirChro will prompt a waiting dialog during the calculation. You can stop the calculation any time by clicking the button "Cancel Task".


Figure 38 Waiting dialog
Figure 38 Waiting dialog


Comparative Distribution Chart

It is interesting to compare the statistics of several networks. These networks can be generated from different organisms, mutation types, or experimental conditions. NetCirChro provides the chart comparison between several networks.


Figure 39 Comparison options
Figure 39 Comparison options


In the comparison option window, you can choose which network will be used for the comparison, and which chart will be displayed. NetCirChro will display the charts for the selected network side-by-side.


Figure 40 Comparison chart
Figure 40 Comparison chart


Cluster Coefficient and Coordinate Closeness

Cluster coefficient defines the connection density in the neighbors of a network node. Coordinate closenenss is defined as the reciprocal of the average gene distance of a gene.

For every node in the network, NetCirChro gets the neighbors of the node and calculates the cluster coefficient and coordinate closeness. The values are plotted as a scatter plot.


Figure 41 Cluster coefficient versus coordinate closeness
Figure 41 Cluster coefficient versus coordinate closeness


NetworkAnalyzer

NetworkAnalyzer is a Cytoscape plugin to calculate many network topological properties. NetCirChro automatically detects if NetworkAnalyzer is installed in the user's Cytoscape folder. If the plugin exists, NetCirChro will add two menus under "Statistics" menu which provides a shortcut to the feature of NetworkAnalyzer plugin.

This NetCirChro version is compatible with NetworkAnalyzer 2.6.1.


Figure 42 Network Analyzer menu
Figure 42 Network Analyzer menu


Help

NetCirChro provides a comprehensive help document with the application. The help system also has the fulltext search capability. You can get the help from the menu "Help | Help Contents".

For downloading the plugin and sample data, please visit http://exon.niaid.nih.gov.

For questions and comments, please email NetCirChroSupport@niaid.nih.gov.

References

Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang J.T., Ramage, D., Amin, N., Schwikowski, B., Ideker, T. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13:2498-2504.

Assenov, Y., Ramírez, F., Schelhorn, S.E., Lengauer, T., Albrecht, M. Computing topological parameters of biological networks. (2008). Bioinformatics. 24(2):282-284.

Citing NetCirChro

In any work or product derived from this material, proper attribution of the authors as the source of the software or data should be made.

Acknowledgements should read:

"The NetCirChro software is developed and maintained at the Bioinformatics and Computational Biosciences Branch (BCBB), Office of Cyber Infrastructure and Computational Biology (OCICB), National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland, United States of America. The software is freely available for download at http://exon.niaid.nih.gov."

License

NetCirChro is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received a copy of the GNU General Public License along with this program; if not, write to the Free Software Foundation, Inc., 59 Temple Place - Suite 330, Boston, MA 02111-1307, USA.

Release Notes

v1.2: Fix importing NCBI data bug using new address

v1.1: Support Cytoscape 2.8.x

v1.0: First release for Cytoscape 2.6.x

Acknowledgements

NetCirChro is built with a number of open source third-party Java libraries. The NetCirChro team gratefully acknowledges the following libraries:

NetCirChro uses a simplified curve generation algorithm derived from CirCos: