# KEGGlincs Workflows

#### Shana White

#### 2021-10-26

##Overview of KEGGlincs

The package KEGGlincs and the functions contained within it are designed such that users can explore KEGG pathways in a more meaningful and informative manner both visually and analytically. This method of pathway analysis is approached via functions that handle the following (related) objectives:

- ‘Expanding’ node mapping for [assumed to be] paralogous node entries and grouped node entries
- Allowing data to be explicitly mapped to ‘expanded’ pathway edges (no summarization necessary)

The idea of ‘expanded’ nodes and edges should become very clear after reviewing the following example KEGGlincs workflows. Please keep in mind, the individual functions detailed in the following workflows are incorporated into the `KEGG_lincs` ‘master function’; these workflows are designed to provide users a with a better understanding of how this function works, how pathway topology is represented in KGML files, and how this package could be used with non-LINCS edge data (see Workflow 2).

##Workflow 1: Visualize detailed ‘expanded’ KEGG pathways ###No data added to edges

This workflow is intended to give users insight into the ‘expansion’ of KEGG pathway mapping via manipulation of the source KGML file. The only input required is the KEGG pathway ID for your pathway of choice. The primary goal for this method of pathway re-generation is to give users insight into the complexity that underlies many KEGG pathways but is in a sense ‘hidden’, yet hard-coded, in the curated KGML files. Users can also see the exact pathway topology that is used for input in analyses such as SPIA (Signaling Pathway Impact Analysis).

####Initialize KEGGlincs package (development phase)

    library(KEGGlincs)

####Download and parse the most current KGML file for Fox0 signaling pathway

    FoxO_KGML <- get_KGML("hsa04068")

    #Information from KGML can be accessed using the following syntax:
    slot(FoxO_KGML, "pathwayInfo")
    ## [ Title ]: FoxO signaling pathway
    ## [ Name ]: path:hsa04068
    ## [ Organism ]: hsa
    ## [ Number ] :04068
    ## [ Image ] :http://www.kegg.jp/kegg/pathway/hsa/hsa04068.png
    ## [ Link ] :http://www.kegg.jp/kegg-bin/show_pathway?hsa04068

####The code chunks below are useful for viewing the original pathway image.

    #Get address for pathway with active links:
    slot(slot(FoxO_KGML, "pathwayInfo"), "image")
    ## [1] "http://www.kegg.jp/kegg/pathway/hsa/hsa04068.png"

    #Download a static pathway image (png file) to working directory:
    image_link <- slot(slot(FoxO_KGML, "pathwayInfo"), "image")
    download.file(image_link, basename(image_link), mode = "wb")

#####Rendering of the png file for the p53 signaling pathway from KEGG:

#####The following commands produce ‘expanded’ node and edge sets Note that KEGG IDs are converted to gene/compound symbols; this conversion accounts for the majority of computing time behind the `expand_KEGG_mappings` function. For quicker map generation, users may chose to change the argument `convert_KEGG_IDs` to `FALSE`; this will result in edges being identified by pairs of accession numbers instead of symbols in the final pathway map (example at end of this workflow using `KEGG_lincs` master function).

    FoxO_KEGG_mappings <- expand_KEGG_mappings(FoxO_KGML)

    FoxO_edges <- expand_KEGG_edges(FoxO_KGML, FoxO_KEGG_mappings)

#####Compare counts for ‘expanded’ vs. ‘unexpanded’ nodes and edges:

    length(graph::nodes(FoxO_KGML)) # 'Un-expanded' nodes
    ## [1] 98
    nrow(FoxO_KEGG_mappings)        # 'Expanded' nodes
    ## [1] 164

    length(graph::edges(FoxO_KGML)) # 'Un-expanded' edges
    ## [1] 78
    nrow(FoxO_edges)                # 'Expanded' edges
    ## [1] 457

#####Add graphing information to nodes and edges and get graph object: Note: While the `node_mapping_info` function is rather trivial, the `edge_mapping_info` functions differently when data is added.

    #Modify existing data sets; specify as nodes and edges
    FoxO_node_mapping_info <- node_mapping_info(FoxO_KEGG_mappings)
    FoxO_edge_mapping_info <- edge_mapping_info(FoxO_edges)

    #Create an igraph object
    GO <- get_graph_object(FoxO_node_mapping_info, FoxO_edge_mapping_info)
    class(GO)
    ## [1] "igraph"

#####Finally, transform graph object and send to Cytoscape with one command:

    cyto_vis(GO, "FoxO Pathway with Expanded Edges[no data added]")

#####Graph rendered in Cytoscape: Edge Color Key:

Red: Activation or Expression *

Orange: Activating PTM **

Green: PTM (no activation/inhibition activity defined)

Blue: Inhibition

Purple: Inhibiting PTM

Black(solid): Binding/Association

Black(dashed): Indirect effect (no activation/inhibition activity defined)

*Any dashed colored line indicates that the effect is indirect

**PTM = post-translational modification or, as KEGG defines them, ‘molecular events’.

- The specific types of PTMS (indicated by edge label) include:
  - +p: phosphorylation
  - -p: dephosphorylation
  - +g: glycosylation
  - +u: ubiquitination
  - +m: methylation

Notice that the original KEGG pathway image includes visual elements such as cellular-component-demarcations and certain edges (especially those ‘connecting’ genes to other pathways) that are not rendered in Cytoscape. These are features that are either not explicitly part of the pathway topology (i.e. not nodes or edges connecting nodes) or have not been hard-coded in the KGML file. The node labels may also differ between maps (KEGGlincs labels nodes as the first ‘alias’ in the respective KGML slot as there is no corresponding ‘label’ slot).

#####Alternative: KEGG_lincs master function The steps above may be avoided if the user does not wish to generate intermediary files/objects by making use of the function `KEGG_lincs` as follows:

    KEGG_lincs("hsa04068")

If users would like the Cytoscape-rendered map along with the detailed list of expanded edges (as an R object), `KEGG_lincs` can be invoked as follows:

    FoxO_edges <- KEGG_lincs("hsa04068")

To speed up the mapping process (at the expense of having edges labelled with pairs of gene accession numbers as opposed to symbols) users may change the default `convert_KEGG_IDs` argumet to `FALSE`

    KEGG_lincs("hsa04068", convert_KEGG_IDs = FALSE)

*Note: As with `cyto_vis`, please use the function `KEGG_lincs_auto` (after sourcing `cyto_vis_auto`) available from : https://github.com/uc-bd2k/KEGGlincs/blob/master/R/KEGG_lincs_auto.R #####Graph rendered in Cytoscape without edge accession numbers converted:

##Workflow 2: Overlay data to edges of KEGG pathway ###Specific use case: LINCS L1000 Knock-out data

While the functions described in Workflow 1 are certainly useful for any users wishing to gain deeper insight into KEGG pathway topology and ‘hard-coded’ KGML information, the drving force motivating the KEGGlincs package development is the association of experimental data with pathway edges.

The companion data package KOdata provides data for the edges rendered by the master function `KEGG_lincs`. This data package includes two unique data sets; one contains lists of significantly up- and down-regulated genes corresponding to knocked-out genes (within individual experiments, genes are ‘turned off’ via shRNA) across a variety of cell-lines measured at specific times and the other is a binary record of baseline gene expression (gene is either expressed or not expressed) for most cell-lines from the knock-out data set.

While this package was developed primarily as a way to compare pathway topology between cell-lines or within cell-lines [across time] using LINCS L1000 data, this workflow will demonstrate the package’s flexibility for users incorporating edge data from any source.

#####Example: Comparing p53 Signalling Pathway between Cell-Lines As a hypothetical scenario, our goal will be to compare pathway topology between cell-lines for an important cancer-related pathway: the p53 Signaling Pathway.

The ‘default’ pathway (with no data added to edges) can be generated either by following Workflow 1 or by using the `KEGG_lincs` master function as follows:

    KEGG_lincs("hsa04115")

From here, the first few lines of code are similar to those of Workflow 1:

    p53_KGML <- get_KGML("hsa04115")
    p53_KEGG_mappings <- expand_KEGG_mappings(p53_KGML)
    p53_edges <- expand_KEGG_edges(p53_KGML, p53_KEGG_mappings)

An important aspect of the L1000 knock-out and expression data is that it is incomplete; experimental data is not uniformly available for each cell-line.
Therefor (for this specific example with this specific data set) it is instructive to find out which cell-lines make sense to compare; intuitively, cell-lines with a similar percentage of pathway genes knocked out would be well suited for comparison. The following command accomplished this task in the form of an easily interpretable graphical output:

    path_genes_by_cell_type(p53_KEGG_mappings)

Another function (`refine_mappings`) is automatically envoked when the `KEGG_lincs` master function is called; this function ‘prunes’ edges from the pathway that would not exist in the cell-line based on baseline expression data. This is beyond the scope of this workflow and will be detailed elsewhere.

If users would like to access lists of specific gene knock-outs within the chosen pathway for each cell-line, the command can be invoked as follows:

    p53_L1000_summary <- path_genes_by_cell_type(p53_KEGG_mappings, get_KOs = TRUE)

Resulting data frame:

| cell_type | num_pathway_KOs | knock_outs | color
--- | --- | --- | --- | ---
2 | A549 | 48 | ATM , BAX , BBC3 , BCL2L1 , BID , CASP3 , CASP9 , CCNB1 , CCNB2 , CCND2 , CCND3 , CCNE1 , CCNE2 , CCNG1 , CCNG2 , CDK1 , CDK2 , CDK4 , CDK6 , CDKN1A , CDKN2A , CHEK2 , CYCS , DDB2 , FAS , GADD45A , GADD45B , IGF1 , IGFBP3 , MDM2 , MDM4 , PMAIP1 , PPM1D , PTEN , RCHY1 , SERPINB5 , SERPINE1 , SESN1 , SFN , SIAH1 , THBS1 , TNFRSF10A, TNFRSF10B, TP53 , ATR , BCL2 , CCND1 , CHEK1 | red
10 | MCF7 | 48 | ATM , BAX , BBC3 , BCL2 , BCL2L1 , BID , CASP3 , CASP8 , CASP9 , CCNB1 , CCNB2 , CCND2 , CCND3 , CCNE1 , CCNE2 , CCNG1 , CCNG2 , CDK1 , CDK2 , CDK4 , CDK6 , CDKN1A , CDKN2A , CHEK1 , CHEK2 , CYCS , DDB2 , FAS , GADD45A , GADD45B , MDM2 , MDM4 , PPM1D , PTEN , RCHY1 , RRM2 , SERPINB5 , SERPINE1 , SFN , SIAH1 , THBS1 , TNFRSF10A, TNFRSF10B, TP53 , TSC2 , ATR , CCND1 , IGF1 | red
8 | HEPG2 | 49 | ATM , ATR , BAX , BBC3 , BCL2 , BCL2L1 , BID , CASP3 , CASP8 , CASP9 , CCNB1 , CCNB2 , CCND1 , CCND2 , CCND3 , CCNE1 , CCNE2 , CCNG1 , CCNG2 , CDK1 , CDK4 , CDK6 , CDKN1A , CDKN2A , CHEK1 , CYCS , DDB2 , GADD45A , GADD45B , IGF1 , MDM2 , PMAIP1 , PPM1D , PTEN , RCHY1 , RRM2 , SERPINB5 , SERPINE1 , SESN1 , SFN , SIAH1 , THBS1 , TNFRSF10A, TNFRSF10B, TP53 , TSC2 , FAS , CDK2 , CHEK2 | red
13 | PC3 | 49 | APAF1 , BAX , BBC3 , BCL2L1 , BID , CASP3 , CASP8 , CCNB1 , CCNB2 , CCND1 , CCND2 , CCND3 , CCNE1 , CCNE2 , CCNG1 , CCNG2 , CDK1 , CDK2 , CDK4 , CDK6 , CDKN1A , CDKN2A , CHEK1 , CYCS , DDB2 , GADD45A , GADD45B , IGF1 , IGFBP3 , MDM2 , MDM4 , PPM1D , PTEN , RCHY1 , RRM2 , SERPINB5 , SESN1 , SIAH1 , THBS1 , TNFRSF10A, TNFRSF10B, TP53 , TSC2 , FAS , SFN , ATM , BCL2 , ATR , CHEK2 | red
1 | A375 | 50 | APAF1 , ATR , BAX , BBC3 , BCL2 , BCL2L1 , BID , CASP3 , CASP8 , CASP9 , CCNB1 , CCNB2 , CCND2 , CCND3 , CCNE1 , CCNG1 , CCNG2 , CDK1 , CDK2 , CDK6 , CDKN1A , CDKN2A , CHEK1 , CHEK2 , DDB2 , FAS , GADD45A , GADD45B , IGFBP3 , MDM2 , MDM4 , PMAIP1 , PPM1D , PTEN , RCHY1 , RRM2 , SERPINB5 , SERPINE1 , SESN1 , SFN , SIAH1 , THBS1 , TNFRSF10A, TNFRSF10B, TP53 , TSC2 , ATM , IGF1 , CDK4 , CCND1 | red
4 | HA1E | 51 | ATR , BAX , BBC3 , BCL2L1 , BID , CASP3 , CASP8 , CASP9 , CCNB1 , CCNB2 , CCND1 , CCND2 , CCND3 , CCNE1 , CCNE2 , CCNG1 , CCNG2 , CDK1 , CDK2 , CDK4 , CDK6 , CDKN1A , CDKN2A , CHEK2 , CYCS , DDB2 , FAS , GADD45A , GADD45B , IGF1 , IGFBP3 , MDM2 , MDM4 , PMAIP1 , PPM1D , PTEN , RCHY1 , RRM2 , SERPINB5 , SERPINE1 , SESN1 , SFN , SIAH1 , THBS1 , TNFRSF10A, TNFRSF10B, TP53 , TSC2 , BCL2 , ATM , CHEK1 | red

The bar plot suggests that the group of cell lines colored in red have similar amounts of pathway information; for this example we will compare the PC3 (prostate cancer) and HA1E (immortalized normal kidney epithelial) cell-lines.

The following commands use the data objects generated above to generate cell-line specific edge attributes corresponding to specific pathway edges and the information from the L1000 knock-out data set:

    p53_PC3_data <- overlap_info(p53_KGML, p53_KEGG_mappings, "PC3")
    ## Number of genes documented in selected pathway = 73
    ## Number of pathway genes in dataset = 49
    ## Coverage = 67.12%

    p53_HA1E_data <- overlap_info(p53_KGML, p53_KEGG_mappings, "HA1E")
    ## Number of genes documented in selected pathway = 73
    ## Number of pathway genes in dataset = 51
    ## Coverage = 69.86%

Example of edge data generated for the PC3 cell line (data columns generated after applying Fisher’s exact test not shown):

| knockout1 | knockout2 | UP | DOWN | UK1_DK2 | DK1_UK2
--- | --- | --- | --- | --- | --- | ---
33 | SIAH1 | CYCS | 0 | 3 | 0 | 1
69 | IGFBP3 | IGF1 | 7 | 7 | 0 | 0
124 | GADD45A | CDK1 | 1 | 2 | 0 | 2
136 | GADD45A | CCNB1 | 7 | 10 | 3 | 0
137 | GADD45A | CCNB2 | 0 | 4 | 1 | 0
169 | GADD45B | CDK1 | 2 | 0 | 1 | 1

The argument `keep_counts_only` in `overlap_info` is by default set to `FALSE`. Users may be interested in follow-up analysis of the genes that are found to be expressed in a concordant/discordant fashion between pairs of knock outs. This information is easily accessible by changing the default as follows:

    p53_PC3_data_with_gene_lists <- overlap_info(p53_KGML, p53_KEGG_mappings,
                                                "PC3", keep_counts_only = FALSE)
    ## Number of genes documented in selected pathway = 73
    ## Number of pathway genes in dataset = 49
    ## Coverage = 67.12%

Example of edge data generated for the PC3 cell line that includes lists of concordant/discordant genes for knock out pairs (count data not shown):

| knockout1 | knockout2 | genes.symbolsUP | genes.symbolsDOWN | genes.symbolsUPK1_DOWNK2 | genes.symbolsDOWNK1_UPK2
--- | --- | --- | --- | --- | --- | ---
33 | SIAH1 | CYCS |  | S100A13, DNM1L , S100P |  | PDHX
69 | IGFBP3 | IGF1 | RRS1 , IGKV1-17 , IGKC , IGKV2D-28, IGKV2-28 , IGKV1D-39, IGKV1-39 | GRN , TIMP1, CDC20, GLOD4, NR2F6, LSM5 , PARP2 |  |
124 | GADD45A | CDK1 | STIMATE | RPS4Y1, POLR2K |  | IQCE , HMGCS1
136 | GADD45A | CCNB1 | SERPINA3, CEBPD , MAL , MAMLD1 , GNAI1 , IGLV3-25, NA | TIMP1 , EXT1 , TRAPPC3 , ZW10 , ALDH7A1 , LDHB , IARS2 , LAP3 , CHMP4A , TNFRSF21 | CLTB, CLTB, CLTB |
137 | GADD45A | CCNB2 |  | RPS4Y1 , MACF1 , KIAA0754, MRPS2 | MBTPS1 |
169 | GADD45B | CDK1 | LAMA3, XIST |  | NCAPH | AARS1

By default, the function `overlap_info` generates edges that are ‘mapped’, i.e. the edge between any two nodes is hard-coded in the KGML file even though data could be generated for all possible (think de novo) edges. A workflow that describes how this can be achieved by simply changing a few default arguments will be added shortly.

Notice the format of the data; in particular that the first two columns contain gene symbols and act as a primary key (unique identifier) for an edge and the rest of the columns are edge attributes. The following function `add_edge_data` can be used with any dataset in this format and will append selected columns to the edge dataset. Note that the data supplied does not need to be pre-arranged in correct source -> target order as specified by the pathway topology; the function automatically re-orients pairs correctly.

    p53_PC3_edges <- add_edge_data(p53_edges, p53_KEGG_mappings,
                                        p53_PC3_data, only_mapped = TRUE,
                                        data_column_no = c(3,10,12))

    ## Number of edges documented in selected pathway = 94
    ## Number of edges with corresponding user data = 62
    ## Coverage = 65.96%

    p53_HA1E_edges <- add_edge_data(p53_edges, p53_KEGG_mappings,
                                    p53_HA1E_data, only_mapped = TRUE,
                                    data_column_no =  c(3,10,12))

    ## Number of edges documented in selected pathway = 94
    ## Number of edges with corresponding user data = 66
    ## Coverage = 70.21%

The following series of commands follow from workflow 1 (with minor adjustments to arguments, notably ensuring that `data_added = TRUE` is specified). The edges in the resulting pathway maps are conditionally formatted to represent both the significance and magnitude of the relationship between corresponding nodes based on their concordance/discordance of up/down-regulated genes as measured by Fisher’s Exact Test.

- Note that [in Cytoscape] graphs rendered in the same session inherit certain style elements from existing graphs that are not updated when the new graph gets pushed (such as range for conditional formatting); therefor it is [in this author’s opinion] best-practice to start with a fresh session when mapping requires conditional formatting.

    p53_node_map <- node_mapping_info(p53_KEGG_mappings)

    p53_edge_map_PC3 <- edge_mapping_info(p53_PC3_edges, data_added = TRUE,
                                            significance_markup = TRUE)
    p53_edge_map_HA1E <- edge_mapping_info(p53_HA1E_edges, data_added = TRUE,
                                            significance_markup = TRUE)

    PC3_GO <- get_graph_object(p53_node_map, p53_edge_map_PC3)
    HA1E_GO <- get_graph_object(p53_node_map, p53_edge_map_HA1E)

    cyto_vis(PC3_GO, "Pathway = p53, Cell line = PC3")
    #Option: Save PC3 as .cys file and start a fresh session in Cytoscape
    cyto_vis(HA1E_GO, "Pathway = p53, Cell line = HA1E")

Edge colors represent the following possible combinations of direction of Fisher’s Exact Test summary scores (a modified Odd’s Ratio score; either positive(+) or negative (-)) and their corresponding [adjusted] p-values:

Red: OR(+), pval(sig)

Orange: OR(+), pval(non-sig)

Purple: OR(-), pval(non-sig)

Blue: OR(-), pval(sig)

#####Graphs rendered in Cytoscape:

Note that as with Workflow 1, the `KEGG_lincs` master function can automatically generate pathway maps identical to the final maps resulting from Workflow 2 as follows:

    KEGG_lincs("hsa04115", "PC3", refine_by_cell_line = FALSE)
    KEGG_lincs("hsa04115", "HA1E", refine_by_cell_line = FALSE)

The only differences are that the `KEGG_lincs` function generates titles with a slightly modified format and automatically ‘refines’ pathway edges for cell-lines with baseline expression data.

#####Graphs rendered in Cytoscape: