Sequnce Similarity Networks Tool

Genome Neighborhood Networks Tool

Result page and file download

EFI-GNT Files and Node Attributes

As described in the previous section, EFI-GNT generates a colored version of the input SSN as well as two formats of genome neighbor networks (GNNs) for download (all three in the xgmml format) and subsequent analysis with Cytoscape.

This section provides a detailed description of the colored SSN and both GNN formats, with emphasis on the node attributes that are provided for the GNNs; these include the query-neighbor distances, co-occurrence frequencies, and the identities of the neighbor’s Pfam family that are used for pathway predictions.

Colored SSN

The colored SSN assists the user in analyzing the GNNs by allowing color-guided association of the cluster nodes in the GNNs with the clusters in the input SSN (Figure 1).

Figure 1. Example of a colored SSN

EFI-GNT assigns a unique number and color to each multi-node cluster in the input SSN. Node attributes are added for the cluster number (Cluster Number) and color (node.fillColor). The clusters are ordered in order of decreasing number of sequences in the clusters (Cluster Sequence Count).

The colored SSN also includes node attributes to indicate whether the sequence has a match in the ENA files (Present in ENA Database?), neighbors in the ENA files (Genome Neighbors in ENA Database?), and the name of the ENA file (ENA Database Genome ID).

In full networks, singletons in the input SSN are excluded from the GNN analysis, while in Rep node networks, singletons with 1 sequence are excluded. Singletons excluded from the GNN analysis will appear in the colored SSN with the Cytoscape default color (cyan). All singletons will not have a cluster number assigned.

Two formats for GNN are generated

EFI-GNT generates the GNN in two formats that provide different query-neighbor perspectives to assist predictions of pathways. The formats differ in the identities of the cluster hub-node (neighbor Pfam family or query SSN cluster) and spoke-nodes (query SSN cluster or Pfam family, respectively).

A GNN contains clusters (hub-node and ≥ 1 spoke‑node) that provide genome neighborhood information (query-neighbor co-occurrence frequencies and distances) for the sequences in the clusters in the input SSN.

In the first GNN format (Figure 2, below), a cluster is present for each query SSN cluster (hub-node, center) that was used to identify genome neighbors (spoke-nodes). This format allows the user to identify functionally linked enzymes, as deduced from genome proximity, that constitute the metabolic pathway in which the sequences in the query SSN cluster participate.

Figure 2. SSN Cluster Hub-Nodes and Pfam Family Spoke-Nodes GNN: The hub-node for each cluster, a hexagon, is the cluster number. The hub-nodes are colored with the unique color assigned for the Colored SSN and labeled with the unique cluster number that was assigned.

In the second GNN format (Figure 3, below), a cluster is present for each Pfam family (hub-node, center) that was identified as a neighbor to queries in the SSN clusters (spoke-nodes). This format allows the user to assess whether queries in multiple SSN clusters are neighbors to members of the same Pfam family and, therefore, may have the same in vitro activities and in vivo metabolic functions.

Figure 3. Pfam Family Hub-Nodes and SSN Cluster Spoke-Nodes GNN: The hub-node, a hexagon colored grey, represents the neighbor Pfam family. These are labeled with the Pfam short name for the family (e.g., DAO for PF01266, FAD-dependent oxidoreductase). For multidomain proteins, the label is a composite of the Pfam short names for the component domains (e.g., FGGY_N- FGGY_C for PF00370-PF02782, the N- and C-terminal domains in the FGGY family of carbohydrate kinases).

In both formats, the node attributes for the Pfam family and SSN cluster nodes contain the same information; these provide information about the query-neighbor relationships that can be used to infer functional relationships (co-occurrence frequency and distance) that enable the prediction of in vitro activities and in vivo metabolic pathways.

If the nodes are not automatically colored when the SSN is opened in Cytoscape, the Style Control Panel can be used to color the nodes in the SSN: In the Fill Color property, select "node:node.fillColor" for the Column value and "Passthrough Mapping" for the Mapping Type value.

Additional Files for Download

In addition to the colored SSN, several text files/folders of text files are available for download. These include:

  1. "UniProt ID-Color-Cluster Number Mapping Table" is a tab-delimited text file with three columns with headers, "UniProt ID", "Cluster Number", and "Cluster Color"; a description of this file and its use was provided in the Color SSN Utility section of the EST Tutorial.
  2. "UniProt IDs per Cluster" is a folder of files for each cluster that list the UniProt IDs for the sequences in the cluster; a description of the files in this folder and their use was provided in the Color SSN Utility section of the EST Tutorial.
  3. "FASTA Files per Cluster" is a folder of files for each cluster that contain the FASTA-formatted sequences for the sequences in the cluster; a description of the files in this folder and their use was provided in the Color SSN Utility section of the EST Tutorial.
  4. "Pfam Neighbor Mapping Tables" is a folder of tab-delimited text files for each neighbor Pfam family that includes the following columns: "Query ID", "Neighbor ID", "Neighbor Pfam", "SSN Query Cluster #", "SSN Query Cluster Color", "Query-Neighbor Distance" (absolute value of distances in ORFs), and "Query-Neighbor Directions" (relative directions of transcription). These files can be used with the Cytoscape BridgeDB App to add these columns as node attributes to the SSN for the neighbor Pfam family so that the neighbors can be located/analyzed in the sequence-function context of the family. The "SSN Query Cluster Color" node attributes can be used with "pass-through mapping" in the Cytoscape Style Color panel to color the nodes in the neighbor Pfam family with the colors in the colored SSN generated by EFI-GNT 1.0, thereby facilitating the determination of whether the neighbors identified in the genome neighborhoods are orthologous. A concatenated file that contains all of the information is also available and may be more convenient for adding node attributes to SSNs for multiple Pfam families.
  5. "Neighbors without PFAM assigned per Cluster" is a folder of tab-delimited text files for each SSN cluster that lists the accession IDs of neighbors not assigned to Pfam families. These files allow SSNs to be generated with Option D of EFI-EST 2.0 so that protein families not curated by Pfam can be identified. The user should use an alignment score of ~20 to filter the SSN; in most cases, this alignment score will segregate the SSN into protein families.
  6. "No Matches/No Neighbors File" is a tab-delimited text file with two columns: "UniProt ID" and "No Match/No Neighbor" ("nomatch" or "neighbor"). The same information is included in the colored SSN generated by EFI-GNT 1.0 (vide infra).

Description of node attributes for the various output files is available below and is provided in two formats: a verbose description and a tabular summary.

Details for format GNN 1: SSN Cluster Hub-Nodes and Pfam Family Spoke-Nodes

In the first GNN format, each hub-node corresponds a cluster present in the SSN (center). The cluster hub-node color and number are identical to those in the colored SSN. Sequences belonging to that cluster were used to retrieve genome neighbors (spoke-nodes; Figure 2). This format allows the user to identify functionally linked enzymes, as deduced from genome proximity, that constitute the metabolic pathway in which the query participates.

A spoke-node, colored grey and represented as a hexagon, is present for each Pfam family that was identified as a neighbor of a query sequence in the cluster represented by the hub-node. These are labeled with the short name for the Pfam family (e.g., Aldedh for PF00171, aldehyde dehydrogenase). For multidomain proteins, the hub-node name is a composite of the short names for the component domains (e.g., HTH_1-LysR for PF00126-PF03466, the N-terminal HTH DNA-binding and C-terminal ligand binding domains of LysR transcriptional regulators). A spoke-node ("none") will be present for neighbors that have not been assigned to a Pfam family.

The size of the spoke-node (node.size) is determined by the value of the Co-occurrence node attribute [decimal value of the ratio of the number of queries (sequences with neighbors) in the cluster that found neighbors in the Pfam family to the number of queriable sequences in the cluster (Queries with Pfam Neighbors/Queriable SSN Sequences); see below]—the larger the co-occurrence frequency of the SSN cluster queries and their genome neighbors, the larger the spoke-node. The value of node.size [calculated as (Co-occurrence * 100)] is used by Cytoscape to draw the node.

The shape of spoke-node (node.shape) is determined by the values of two node attributes for the neighbors that were identified (in the Query-Neighbor Accession node attribute): a triangle if a SwissProt description is available, a square if a Protein Data Bank (PDB) code is available; a diamond if both an EC number and a PDB are available; or a circle if neither an EC number or a PDB code is available. The availability of a SwissProt description and/or a PDB code suggests that the function of the neighbor may be known. The node shape in the node shape node attribute (node.shape) is used by Cytoscape to draw the node. The network can be filtered with the Select Panel to select specific shapes, i.e. different levels of confidence about the functions of the neighbors.

The identities of the neighbors not associated with a Pfam family (no Pfam or "none" family) also are provided in the Neighbor Accession and Query-Neighbor Arrangement node attributes (described in detailed in the descriptions of the node attributes).

Node attributes for each SSN cluster hub-node

shared name (generated by Cytoscape): the unique number assigned to each cluster in the input SSN (singletons are not included) This node attribute is text—strings of characters can be selected with the Select Control Panel.
name (generated by Cytoscape): the unique number assigned to each cluster in the input SSN (singletons are not included) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Cluster Number: the unique number assigned to each cluster in the input SSN (singletons are not included) This node attribute has a numerical value— a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Sequences in SSN Cluster: The total number of sequences in the SSN cluster. This node attribute has a numerical value—a specific Cluster Number or a range of Cluster Numbers can be selected with the Select Control Panel.
# of Sequences in SSN Cluster with Neighbors: The number of sequences in the SSN cluster that have genome neighbors in the bacterial and fungal ENA sequence files (queriable sequences). The value of this node attribute is calculated by:

Total number of sequences in the SSN cluster (# of Sequences in SSN Cluster) –
number of sequences that did not have a match in the ENA sequence files (list provided in the nomatch file that can be downloaded) –
number of sequences for which the ENA sequence files did not provide genome neighborhoods (list provided in the noneighbor file that can be downloaded).

This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
Hub Queries with Pfam Neighbors: A summary for all spoke-node Pfam families of the number of queriable sequences in the hub-node SSN cluster (# of Sequences in SSN Cluster with Neighbors) for which a neighbor in the Pfam family was found in the following format:

cluster#:Pfam#:#Queries with Pfam Neighbors

where
  • "cluster#" is the cluster# for the query
  • "Pfam#" is the spoke-node Pfam family number
  • "#Queries with Pfam Neighbors" is the number of queriable sequences in the SSN cluster (# of Sequences in SSN Cluster with Neighbors) for which a neighbor in the spoke-node Pfam family was found.
A query may find multiple members of the Pfam family, but this node attribute reports only the number of queries that found any neighbor in the Pfam family. The neighbors in the Pfam family need not be orthologues (share the same function). This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Hub Pfam Neighbors: A summary for all spoke-node Pfam families of the number of neighbors in the Pfam family that were found by the queries in the hub-node SSN cluster in the following format:

cluster#:Pfam#:#Pfam Neighbors

where
  • "cluster#" is the cluster# for the query
  • "Pfam#" is the spoke-node Pfam family
  • "#Pfam Neighbors" is the number of neighbors in the spoke-node Pfam family found by the queries in hub-node SSN cluster.
The value of "#Pfam Neighbors" will be greater than the value of the Hub Queries with PFAM Neighbors (previous) node attribute if a query found more than one neighbor in the Pfam family. Again, the neighbors in the Pfam family need not be orthologues (share the same function). This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Hub Average and Median Distances: A summary for all spoke-node Pfam families of the values of the Average Distance and Median Distance node attributes in the following format:

cluster#:"Pfam#:average absolute value of distances:median absolute value of distances

where
  • "cluster#" is the cluster# for the query
  • "Pfam#" is the Pfam# for the neighbor
  • "average absolute value of distances" is the average of the absolute values of distances between the hub-node queries and spoke-node neighbors
  • "median absolute value of distances" is the median absolute value of distances between the hub-node queries and spoke-node neighbors.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Hub Co-occurrence and Ratio: A summary for all spoke-node Pfam families of the values for Co-occurrence and Co-occurrence Ratio node attributes in the following format:

cluster#:Pfam#:Co-occurrence:Co-occurrence Ratio

where
  • "cluster#" is the cluster# for the query
  • "Pfam#" is the Pfam# for the neighbor
  • "Co-occurrence" is the decimal value of the ratio of the number queries that found neighbors in the Pfam family to the number of queriable sequences in the hub-node SSN query cluster (# of Queries with Pfam Neighbors/# of Sequences in SSN Cluster with Neighbors)
  • "Co-occurrence Ratio" is the numerical ratio of the number of queries that found neighbors in the Pfam family to the number of queriable sequences in the hub-node SSN cluster.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Node.fillColor: the hexadecimal number of the unique color assigned to each cluster in the input SSN (singletons are not included). This number is used by the pass-through mapping "Fill Color" style of Cytoscape to color the nodes in the network.

If the nodes are not automatically colored when the SSN is opened in Cytoscape, the Style Control Panel can be used to color the nodes in the SSN: In the Fill Color property, select "node:node.fillColor" for the Column value and "Passthrough Mapping" for the Mapping Type value.

Node.shape: hexagon (used by Cytoscape but can be used in searches to select hub-nodes)
Node.size: 70.0 (used by Cytoscape)
Pfam: empty (a spoke-node attribute)
Pfam description: empty (a spoke-node attribute)
# of Queries with Pfam Neighbors: empty (a spoke-node attribute)
# of Pfam Neighbors: empty (a spoke-node attribute)
Query-Accessions: empty (a spoke-node attribute)
Query-Neighbor Accessions: empty (a spoke-node attribute)
Query-Neighbor Arrangement: empty (a spoke-node attribute)
Average Distance: empty (a spoke-node attribute)
Median Distance: empty (a spoke-node attribute)
Co-occurrence: empty (a spoke-node attribute)
Co-occurrence Ratio: empty (a spoke-node attribute)

Node attributes for each Pfam family spoke-node

shared name: the Pfam family short name or hyphen-separated short names for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel.
name: the Pfam family short name (hyphen-separated names for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel [The shared name and name node attributes are redundant and required by Cytoscape.]
Cluster Number: the hub-node SSN cluster that found neighbors in the Pfam family spoke-node.
Pfam: the Pfam family number (PFxxxxx) (or hyphen-separated numbers for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Pfam description: the Pfam family long name (or hyphen-separated names for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel.
# of Sequences in SSN Cluster with Neighbors: The number of sequences in the SSN cluster that have genome neighbors in the bacterial and fungal ENA sequence files (queriable sequences). The value of this node attribute is calculated by:

Total number of sequences in the SSN cluster (# of Sequences in SSN Cluster) –
number of sequences that did not have a match in the ENA sequence files (list provided in the nomatch file that can be downloaded) –
number of sequences for which the ENA sequence files did not provide genome neighborhoods (list provided in the noneighbor file that can be downloaded).

This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Queries with Pfam Neighbors: The total number of queriable sequences in the hub-node SSN cluster (# of Sequences in SSN Cluster with Neighbors in SSN cluster hub-node) for which any neighbor in this Pfam family was found. A query may find multiple members of the Pfam family, but this node attribute reports only the number of queries that found any neighbor.
# of Pfam Neighbors: The total number of neighbors in the Pfam family found by the queries in the spoke-node SSN cluster. This value of this node attribute will be greater than the value of the Queries with PFAM Neighbors (previous) node attribute if a query found more than one neighbor in the Pfam family. The neighbors in the Pfam family need not be orthologues (share the same function)—this can be evaluated by mapping the neighbors to the SSN for the Pfam family using the spreadsheet/custom node attribute files that can be downloaded. This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
Query-Accessions: A list of the query accession IDs in the SSN cluster that found neighbors.
Query-Neighbor Accessions: Information about the query-neighbor pairs in the Pfam family in the following format:

Pfam#:Query ID:Neighbor ID:EC#:NeighborPDB:ClosestPDB:PDB-E-value:Status

where:
  • "Query ID" is the query accession ID,
  • "Pfam#" is the Pfam# for the neighbor,
  • "Neighbor ID" is the neighbor accession ID,
  • "EC#" is the E.C. number, if any, assigned to the neighbor in the UniProt database,
  • "NeighborPDB is the Protein Databank (PDB) identifier for the neighbor is one is available,
  • "ClosestPDB" is the Protein Databank (PDB) identifier for the most similar sequence to the neighbor with a structure in the PDB database,
  • "PDB-E-value" is the BLAST e-value for the neighbor-ClosestPDB pair, and
  • "Status" (SwissProt/TrEMBL) reports if the in vitro activity of the neighbor has been reviewed by SwissProt.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
[ClosestPDB:PDB-E-value is a novel Node Attribute that indicates whether a sequence shares significant (e-value < e-30) homology with a protein for which an X-ray crystal structure has been deposited in the PDB. The format of this information is "PDB code:e‑value". This information is valuable to computational chemists wanting to construct a structure model using a known structure as a template from a protein similar in sequence. Ideally, the neighbor sequence itself would have a deposited X-ray crystal structure (previous field in this node attribute), but this is most often not the case. Nonetheless, confident structure models have been employed successfully in pathway docking to determine the substrates of unknown enzymes.]
Query-Neighbor Arrangement: Genome context information for neighbors in the Pfam family in the following format:

Pfam#:Query ID:normal/complement:Neighbor ID: noncomplement/complement:Distance

where
  • "Pfam#" is the Pfam# for the neighbor,
  • "Query ID" is the query accession ID,
  • "normal/complement" is the direction of transcription of the gene encoding the query (from the ENA sequence file),
  • "Neighbor ID" is the neighbor accession ID,
  • "normal/complement" is the direction of transcription of the gene encoding the query (from the ENA sequence file), and
  • "Distance" is the distance in orfs between the genes encoding the query and neighbor.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Average Distance: The average of the absolute values of the distances between the queries in the hub-node SSN cluster and their neighbors in the Pfam family. This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
Median Distance: The median value of the absolute values of the distances between the queries in the hub-node SSN cluster and their neighbors in the Pfam family. This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
Co-occurrence: The decimal value of the ratio of the number of queries in the hub-node SSN cluster that found neighbors in the Pfam family to the number of queriable sequences in the SSN cluster (# of Queries with Pfam Neighbors/# of Sequences in SSN Cluster with Neighbors). This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
Co-occurrence Ratio: The numerical ratio of the number of queries in the hub- node SSN cluster that found neighbors in the Pfam family to the number of queriable sequences in the SSN cluster (Hub Queries with Pfam Neighbors/# of Sequences in SSN Cluster with Neighbors). This node attribute is text-strings of characters can be selected with the Select Control Panel.
Node.fillColor: #EEEEEE (grey in hexadecimal); used by Cytoscape. This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.shape: ellipse, diamond, or square (explained above); used by Cytoscape but can be used in searches. This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.size: calculated as (Co-occurrence * 100); used by Cytoscape.
This node attribute is text—strings of characters can be selected with the Select Control Panel.
# of Sequences in SSN Cluster: empty (a hub-node attribute)
# of Sequences in SSN Cluster with Neighbors: empty (a hub-node attribute)
Hub Queries with Pfam Neighbors: empty (a hub-node attribute)
Hub Pfam Neighbors: empty (a hub-node attribute)
Hub Average and Median Distances: empty (a hub-node attribute)
Hub Co-occurrence and Ratio: empty (a hub-node attribute)

Details for GNN format 2: Pfam Family Hub-Nodes and SSN Cluster Spoke-Nodes

In the second GNN format, a cluster is present for each Pfam family (hub-node) that was identified as a neighbor to queries in the SSN clusters (spoke-nodes; Figure 3). This format allows the user to assess whether queries in multiple SSN clusters are neighbors to members of the same Pfam family and, therefore, may have the same in vitro activities and in vivo metabolic functions.

A spoke-node is present for each SSN cluster that identified a member of the Pfam family. The color of the spoke-node is the unique color assigned in the colored SSN; its label is the unique cluster number.

The size of the spoke-node (node.size) is determined by the value of the Co-occurrence node attribute [decimal value of the ratio of the number of queries in the cluster that found neighbors in the Pfam family to the number of queriable sequences in the cluster (# of Queries with Pfam Neighbors/# of Queriable SSN Sequences); see below]—the larger the co-occurrence frequency of the SSN cluster queries and their genome neighbors, the larger the spoke-node. The value of node.size [calculated as (Co-occurrence * 100)] is used by Cytoscape to draw the node.

The shape of spoke-node (node.shape) is determined by the values of two node attributes for the neighbors that were identified (in the Query-Neighbor Accession node attribute): a triangle if a SwissProt description is available, a square if a Protein Data Bank (PDB) code is available; a diamond if both an EC number and a PDB are available; or a circle if neither an EC number or a PDB code is available. The availability of a SwissProt description and/or a PDB code suggests that the function of the neighbor may be known. The node shape in the node shape node attribute (node.shape) is used by Cytoscape to draw the node. The network can be filtered with the Select Panel to select specific shapes, i.e. different levels of confidence about the functions of the neighbors.

The network can be filtered with the Control Select Panel to select specific node shapes, i.e., different levels of confidence about the functions of neighbors.

The identities of the neighbors not associated with a Pfam family are provided in the Neighbor Accession and Query-Neighbor Arrangement node attributes for the Pfam family nodes (described in detailed in the descriptions of the node attributes).

Node attributes for Pfam family hub-nodes

shared name: the Pfam family short name (or hyphen-separated short names for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel.
name: the Pfam family short name (hyphen-separated names for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel. [The shared name and name node attributes are redundant and required by Cytoscape.]
Pfam: the Pfam family number (PFxxxxx) (or hyphen-separated numbers for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Pfam description: the Pfam family long name (or hyphen-separated names for multidomain proteins) This node attribute is text—strings of characters can be selected with the Select Control Panel.
# of Sequences in SSN Cluster: The total number of sequences in the spoke-node SSN clusters. This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Sequences in SSN Cluster with Neighbors: The total number of sequences in the spoke-node SSN clusters that have genome neighbors in the bacterial and fungal ENA sequence files (queriable sequences). The value of this node attribute is calculated by:

Total number of sequences in the spoke-node SSN clusters (# of Sequences in SSN Cluster) –
number of sequences that did not have a match in the ENA sequence files (list provided in the nomatch file that can be downloaded) –
number of sequences for which the ENA sequence files did not provide genome neighborhoods (list provided in the noneighbor file that can be downloaded).

This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Queries with Pfam Neighbors: The total number of queriable sequences in the spoke-node SSN clusters for which a neighbor in the hub-node Pfam family was found. A query may find multiple members of the Pfam family, but this node attribute reports only the number of queries that found any neighbor in the Pfam family. The neighbors in the Pfam family need not be orthologues (share the same function)—this can be evaluated by mapping the neighbors to the SSN for the Pfam family using the spreadsheet/custom node attribute files that can be downloaded. This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Pfam Neighbors: The total number of neighbors in the Pfam family found by the queries in the spoke-node SSN clusters. This value of this node attribute will be greater than the value of the # of Queries with Pfam Neighbors (previous) node attribute if a query found more than one neighbor in the Pfam family. Again, the neighbors in the Pfam family need not be orthologues (share the same function)—this can be evaluated by mapping the neighbors to the SSN for the Pfam family using the spreadsheet/custom node attribute files that can be downloaded. This node attribute has a numerical value—a specific number of neighbors or a range of neighbors can be selected with the Select Control Panel.
Query Accessions: A summary of queries for all spoke-node SSN clusters in the following format:

cluster#:Query ID

where
  • "cluster#" is the cluster# for the query, and
  • "Query ID" is the query accession ID.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Query-Neighbor Accessions: A summary for all spoke-node SSN clusters of information about the query-neighbor pairs in the Pfam family in the following format:

cluster#:Query ID:Neighbor ID:EC#:NeighborPDB:ClosestPDB:PDB-E-value:Status

where
  • "cluster#" is the cluster# for the query,
  • "Query ID" is the query accession ID,
  • "Neighbor ID" is the neighbor accession ID,
  • "EC# "is the E.C. number, if any, assigned to the neighbor in the UniProt database,
  • "NeighborPDB" is the Protein Databank (PDB) identifier for the neighbor is one is available,
  • "ClosestPDB" is the Protein Databank (PDB) identifier for the most similar sequence to the neighbor with a structure in the PDB database,
  • "PDB-E-value" is the BLAST e-value for the neighbor-ClosestPDB pair, and
  • "Status" (SwissProt/TrEMBL) reports if the in vitro activity of the neighbor has been reviewed by SwissProt.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
[ClosestPDB:PDB-E-value is a novel Node Attribute that indicates whether a sequence shares significant (e-value < e-30) homology with a protein for which an X-ray crystal structure has been deposited in the PDB. The format of this information is "PDB code:e-value". This information is valuable to computational chemists wanting to construct a structure model using a known structure as a template from a protein similar in sequence. Ideally, the neighbor sequence itself would have a deposited X-ray crystal structure (previous field in this node attribute), but this is most often not the case. Nonetheless, confident structure models have been employed successfully in pathway docking to determine the substrates of unknown enzymes.]
Query-Neighbor Arrangement: A summary for all spoke-node SSN clusters of genome context information for the neighbors in the Pfam family in the following format:

cluster#:Query ID:normal/complement:Neighbor ID: normal/ complement:Distance

where
  • "cluster#" is the cluster# for the query,
  • "Query ID" is the query accession ID,
  • "normal/complement" is the direction of transcription of the gene encoding the query (from the ENA sequence file),
  • "Neighbor ID" is the neighbor accession ID,
  • "normal/complement" is the direction of transcription of the gene encoding the query (from the ENA sequence file), and
  • "Distance" is the distance in orfs between the genes encoding the query and neighbor.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Hub Average and Median Distances: A summary for all spoke-node SSN clusters of the values of the Average Distance and Median Distance node attributes in the following format:

cluster#:Average Distance:Median Distance

where
  • "cluster#" is the cluster# for the query,
  • "average absolute value of distances" is the average distance between the queries and neighbors in the cluster, and
  • "median absolute value of distances" is the median distance between the queries and neighbors in the cluster.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Hub Co-occurrence and Ratio: A summary for all spoke-node SSN clusters of the values for Co-occurrence and Co-occurrence Rationode attributes in the following format:

cluster#:Co-occurrence:Co-occurrence Ratio

where
  • "cluster#" is the cluster# for the query,
  • "Co-occurrence" is the decimal value of the ratio of the number of queries that found neighbors in the Pfam family to the number of queriable sequences in the cluster (# of Queries with Pfam Neighbors/# of Sequences in SSN Cluster with Neighbors), and
  • "Co-occurrence Ratio" is the ratio of the number of queries that found neighbors in the Pfam family to the number of queriable sequences in the cluster.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Node.fillColor: #FFFFFF (white in hexadecimal; used by Cytoscape) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.shape: hexagon (used by Cytoscape but can be used in searches to select types of nodes) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.size: 70.0 (used by Cytoscape) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Cluster Number: empty (a spoke-node attribute)
Average Distance: empty (a spoke-node attribute)
Median Distance: empty (a spoke-node attribute)
Co-occurrence: empty (a spoke-node attribute)
Co-occurrence Ratio: empty (a spoke-node attribute)

Node attributes for SSN cluster spoke-nodes

shared name: the unique number assigned to each cluster in the input SSN (singletons are not included) This node attribute is text—strings of characters can be selected with the Select Control Panel.
name: the unique number assigned to each cluster in the input SSN (singletons are not included) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Cluster Number: the unique number assigned to each cluster in the input SSN (singletons are not included) This node attribute has a numerical value—a specific Cluster Number or a range of Cluster Numbers can be selected with the Select Control Panel.
# of Sequences in SSN Cluster: The total number of sequences in the SSN cluster. This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Sequences in SSN Cluster with Neighbors: The number of sequences in the SSN cluster that have genome neighbors in the bacterial and fungal ENA sequence files. The value of this node attribute is calculated by:

Total number of sequences in the cluster (# of Total SSN Sequences) –
number of sequences that did not have a match in the ENA sequence files (list provided in the nomatch file that can be downloaded) –
number of sequences for which the ENA sequence files did not provide genome neighborhoods (list provided in the noneighbor file that can be downloaded).

This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Queries with Pfam Neighbors: The total number of queriable sequences (value of # Queriable SSN Sequences) in the SSN cluster for which a neighbor in the hub- node Pfam family was found. A query may find multiple members of the Pfam family, but this node attribute reports only the number of queries that found any neighbor in the Pfam family. The neighbors in the Pfam family need not be orthologues (share the same function)—this can be evaluated by mapping the neighbors to the SSN for the Pfam family using the spreadsheet/custom node attribute files that can be downloaded.
This node attribute has a numerical value—a specific number of sequences or a range of sequences can be selected with the Select Control Panel.
# of Pfam Neighbors: The total number of neighbors in the Pfam family that were found by the sequences in the SSN cluster. This value of this node attribute will be greater than the value of the # Queries with PFAM Neighbors (previous) node attribute if a query found more than one neighbor in the Pfam family. Again, the neighbors in the Pfam family need not be orthologues (share the same function)— this can be evaluated by mapping the neighbors to the SSN for the Pfam family using the spreadsheet/custom node attribute files that can be downloaded. This node attribute has a numerical value—a specific number of neighbors or a range of neighbors can be selected with the Select Control Panel.
Query-Neighbor Accessions: A list of information about the query-neighbor pais in the Pfam family in the following format:

Query ID:Neighbor ID:EC#:Neighbor PDB:Closest PDB:PDB-E-value:Status

where
  • "Query ID" is the query UniProt accession ID,
  • "Neighbor ID" is the neighbor UniProt accession ID,
  • "EC#" is the E.C. number, if any, assigned to the neighbor in the UniProt database,
  • "Neighbor PDB" is the Protein Databank (PDB) identifier for the neighbor if one is available,
  • "ClosestPDB" is the Protein Databank (PDB) identifier for the most similar sequence to the neighbor with a structure in the PDB database,
  • "PDB-E-value" is the BLAST e-value for the neighbor-ClosestPDB pair, and
  • "Status" (Reviewed/Unreviewed) reports if the in-vitro activity of the neighbor has been reviewed by SwissProt.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Query-Neighbor Arrangement: A list of genome context information for the neighbors in the Pfam family in the following format:

Query ID:normal/complement:Neighbor ID:normal/complement:Distance

where
  • "Query ID" is the query UniProt accession ID,
  • "normal/complement" is the direction of transcription of the gene encoding the query (from the ENA sequence file),
  • "Neighbor ID" is the neighbor UniProt accession ID,
  • "normal/complement" is the direction of transcription of the gene encoding the query (from the ENA sequence file), and
  • "Distance" is the distance in orfs between the genes encoding the query and neighbor.
This node attribute is text—strings of characters can be selected with the Select Control Panel. By right clicking on the node attribute, the entries can be copied and pasted into Excel or a text file for further analyses. In Excel, the colon-delimited entries can be easily separated into separate columns.
Average Distance: The average of the absolute values of the distances between the queries in the cluster and their neighbors in the Pfam family. This node attribute has a numerical value—a specific distance or a range of distances can be selected with the Select Control Panel.
Median Distance: The median value of the absolute values of the distances between the queries in the cluster and their neighbors in the Pfam family This node attribute has a numerical value—a specific distance or a range of distances can be selected with the Select Control Panel.
Co-occurrence: The decimal value of the ratio of the number of queries that found neighbors in the Pfam family to the number of queriable sequences in the cluster. This node attribute has a numerical value—a specific co-occurrence or a range of co-occurrences can be selected with the Select Control Panel.
Co-occurrence Ratio: The ratio of the number of queries that found neighbors in the Pfam family to the number of queriable sequences in the cluster. This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.fillColor: the hexadecimal number of the unique color assigned to each cluster in the input SSN (singletons are not included). This number is used by the pass-through mapping "Fill Color" style of Cytoscape to color the nodes in the network. This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.shape: ellipse, diamond, triangle, or square (explained above; used by Cytoscape but can be used in searches to select hub-nodes) This node attribute is text—strings of characters can be selected with the Select Control Panel.
Node.size: calculated as (Co-occurrence * 100) is used by Cytoscape to draw the node. This node attribute is text—strings of characters can be selected with the Select Control Panel.
Pfam: empty (a hub-node attribute)
Pfam description: empty (a hub-node attribute)
Hub Average and Median Distance: empty (a hub-node attribute)
Hub Co-occurrence and Co-occurrence Ratio: empty (a hub-node attribute)

Tabular Summary of Node Attributes

Colored SSN

Node Attribute Description - Options A, B, C with FASTA header reading, D
Name Full network - UniProt accession; Rep Node network - UniProt accession for the longest sequence in the representative node (seed sequence for CD-Hit)
Shared name Full network - UniProt accession; Rep Node network - UniProt accession for the longest sequence in the representative node (seed sequence for CD-Hit)
Number of IDs in Rep Node1 Number of UniProt IDs in the representative node
List of IDs in Rep Node1 List of UniProt IDs in the representative node
Sequence Source Options B, C, and D, “USER” if from user-supplied file, “FAMILY” if from user-specified Pfam/InterPro family, “USER+FAMILY” if from both
Query IDs Option C with FASTA header reading and Option D, Input Query ID(s) that identified a UniProt match
Other IDs Option C with FASTA header reading, additional IDs in headers for a FASTA sequence that did not identify a UniProt match (NCBI BLAST files)
Cluster Number Number assigned to cluster, in order of decreasing number of sequences in the clusters (“999999” for singletons)
Cluster Sequence Count Number of sequences in the cluster
Node.fillColor Unique color assigned to cluster, in hexadecimal
Present in ENA Database? “true” if UniProt ID was found in an ENA file (see ENA Database Genome ID); otherwise “false”
Genome Neighbors in ENA Database? “true” if ENA file has sequences for query plus neighbors; “false” if ENA file has no neighbors; “n/a” if not present in ENA database
ENA Database Genome ID ENA file used to obtain genome neighbors
Organism organism genus/genera and species, from UniProt taxonomy.xml
Taxonomy ID NCBI taxonomy identifier(s), from UniProt
UniProt Annotation Status SwissProt - manually annotated; TrEMBL - automatically annotated; from UniProt
Description protein name(s)/annotation(s), from UniProtKB
SwissProt Description protein name(s)/annotation(s), from UniProtKB for SwissProt reviewed entries
Sequence Length number(s) of amino acid residues, from UniProt
Gene name gene name(s)
NCBI IDs RefSeq/GenBank IDs and GI numbers, from UniProt idmapping
Superkingdom domain of life of the organism, from UniProt taxonomy.xml
Kingdom kingdom of the organism, from UniProt taxonomy.xml
Phylum Phylogenetic phylum of the organism, from UniProt taxonomy.xml
Class Phylogenetic class of the organism, from UniProt taxonomy.xml
Order Phylogenetic order of the organism, from UniProt taxonomy.xml
Family Phylogenetic family of the organism, from UniProt taxonomy.xml
Genus Phylogenetic genus of the organism, from UniProt taxonomy.xml
Species Phylogenetic species of the organism, from UniProt taxonomy.xml
EC EC number, from UniProt
PFAM Pfam family, from UniProt
IPRO InterPro family, from UniProt
PDB Protein Data Bank entry, from UniProt
BRENDA ID BRENDA Database ID, from UniProt
CAZY Name Carbohydrate-Active enZYmes (CAZy) family name(s), from UniProt
GO Term Gene Ontology classification(s), from UniProt
KEGG ID KEGG Database ID, from UniProt
PATRIC ID PATRIC Database ID, from UniProt
STRING ID STRING Database ID, from UniProt
HMP Body Site location(s) of organism(s) in/on the body, if human microbiome organism, spreadsheet from HMP
HMP Oxygen oxygen requirement(s), if human microbiome organism, spreadsheet from HMP
P01 gDNA availability of gDNA(s) at EFI Protein Core, in-house
1 - These attributes are present only in Rep Node Networks


Node Attribute Description - Option C without FASTA header reading
Name zzznnn, where nnn = number of the sequence in FASTA file
Shared Name zzznnn, where nnn = number of the sequence in FASTA file
Description FASTA Header
Sequence Length Length of sequence in FASTA entry
Sequence Source “USER” if from user-supplied file, “FAMILY” if from user-specified Pfam/InterPro family, “USER+FAMILY” if from both
Cluster Number Number assigned to cluster, in order of decreasing number of sequences in the clusters (“999999” for singletons)
Cluster Sequence Count Number of sequences in the cluster
Node.fillColor Unique color assigned to cluster, in hexadecimal
Present in ENA Database? “false”
Genome Neighbors in ENA Database? “n/a”
ENA Database Genome ID none

SSN Cluster Hub-Nodes and Pfam Family Spoke-Nodes

Node Attribute Description - SSN cluster hub-nodes
Shared name Input SSN cluster number
Name Input SSN cluster number
Cluster Number Input SSN cluster number
# of Sequences in SSN Cluster Total number of sequences in SSN cluster
# of Sequences in SSN Cluster with Neighbors Number of sequences in SSN cluster with neighbors (queriable sequences)
Hub Queries with Pfam Neighbors Summary of number of queriable sequences with a neighbor in the Pfam family
Hub Pfam Neighbors Summary of the total # of Pfam neighbors found by the queriable sequences
Hub Average and Median Distances Summary of average and median distances between the query and neighbors in each Pfam family
Hub Co-occurrence and Ratio Summary of the query-neighbor co-occurrence (decimal value) and ratio (fraction) for each Pfam family
Node.fillColor Hexadecimal color for the SSN cluster in the colored SSN, used by Cytoscape
Node.shape "hexagon", used by Cytoscape
Node Size "70.0", used by Cytoscape


Node Attribute Description - Pfam family spoke-nodes
Shared name Pfam family short name
Name Pfam family short name
SSN Cluster Number SSN Cluster that found neighbors in the Pfam family
Pfam Pfam family number (PFnnnnn)
Pfam description Pfam family description
# of Queries with Pfam Neighbors Number of queriable sequences with a neighbor in the Pfam family
# of Pfam Neighbors Number of Pfam neighbors found by the queriable sequences
Query-Accessions List of SSN cluster queries that found neighbors in the Pfam family
Query-Neighbor Accessions Information about query-neighbor pairs in the Pfam family
Query-Neighbor Arrangement Genome context information for the query-neighbor pairs in the Pfam family
Average Distance Average distance (in ORFs) between the SSN cluster queries and Pfam neighbors
Median Distance Median distance (in ORFs) between the SSN cluster queries and Pfam neighbors
Co-occurrence Decimal value of ratio of queries that found neighbors to queriable sequences
Co-occurrence Ratio Ratio of queries that found neighbors to queriable sequences
Node.fillColor #EEEEEE, grey in hexadecimal, used by Cytoscape
Node.shape "ellipse", "diamond", or "square"; explained in on-line tutorial, used by Cytoscape
Node.size Co-occurrence * 100, used by Cytoscape

Pfam Family Hub-Nodes and SSN Cluster Spoke-Nodes

Node Attribute Description - Pfam family hub-nodes
Shared name Pfam family short name
Name Pfam family short name
Pfam Pfam family number (PFnnnnn)
Pfam description Pfam family description
# of Sequences in SSN Cluster Total number of sequences in SSN cluster
# of Sequences in SSN Cluster with Neighbors Number of sequences in SSN cluster with neighbors (queriable sequences)
# of Queries with Pfam Neighbors Number of queriable sequences with a neighbor in the Pfam family
# of Pfam Neighbors Number of Pfam neighbors found by the queriable sequences
Query-Neighbor Accessions Information about query-neighbor pairs in the Pfam family
Query-Neighbor Arrangement Genome context information for the query-neighbor pairs in the Pfam family
Hub Average and Median Distances Summary of average and median distances between the query and neighbors
Hub Co-occurrence and Ratio Summary of the query-Pfam family co-occurrence (decimal value) and ratio (fraction)
Node.fillColor “#FFFFFF”, white in hexadecimal, used by Cytoscape
Node.shape “hexagon”, used by Cytoscape
Node.size “70.0”, used by Cytoscape


Node Attribute Description - SSN cluster spoke-nodes
Shared name Input SSN cluster number
Name Input SSN cluster number
Cluster Number Input SSN cluster number
# of Sequences in SSN Cluster Total number of sequences in SSN cluster
# of Sequences in SSN Cluster with Neighbors Number of sequences in SSN cluster with neighbors (queriable sequences)
# of Queries with Pfam Neighbors Number of queriable sequences with a neighbor in the Pfam family
# of Pfam Neighbors Number of Pfam neighbors found by the queriable sequences
Query-Accessions List of queries in each SSN cluster that found neigbhors in the Pfam family
Query-Neighbor Accessions Information about query-neighbor pairs in the Pfam family
Query-Neighbor Arrangement Genome context information for the query-neighbor pairs in the Pfam family
Average Distance Average distance (in ORFs) between the SSN cluster queries and Pfam neighbors
Median Distance Median distance (in ORFs) between the SSN cluster queries and Pfam neighbors
Co-occurrence Decimal value of ratio of queries that found neighbors to queriable sequences
Co-occurrence Ratio Ratio of queries that found neighbors to queriable sequences
Node.fillColor Hexadecimal color for the SSN cluster in the colored SSN, used by Cytoscape
Node.shape “ellipse”, “diamond”, or “square”; explained in on-line tutorial, used by Cytoscape
Node.size Co-occurrence * 100, used by Cytoscape

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