PancCanNet - Workflow 2

General instructions (read before running the code snippets)

In this second workflow, we will create a protein-protein interaction network of the up- and down-regulated genes in NanoString dataset (Folfirinox treatment). Afterwards, we will extend the network with gene-pathway associations to see in which pathways the differentially expressed genes are present in.

• The script contains several code snippets which should be run one after the other.
• Make sure all the required packages are installed beforehand (BiocManager::install(…)).
• Make sure you have Cytoscape installed (version 3.8.0+) and running before you start running the script.

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Setup

Loading libraries

options(connectionObserver = NULL)

library(dplyr)
library(rWikiPathways)
library(RCy3)
library(RColorBrewer)
library(rstudioapi)
library(readr)

setwd(dirname(getActiveDocumentContext()$path))

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Load differential gene expression dataset

Make sure you ran workflow 1 beforehand, so the differential gene expression file has been generated.

We take the series 1 from the following dataset (NHBE mock treated versus SARS-CoV-2 infected): * https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147507

which is related to the following publication:

Blanco-Melo, Daniel, et al. “Imbalanced host response to SARS-CoV-2 drives development of COVID-19.” Cell 181.5 (2020): 1036-1045.

dataset <- read.csv("data/DEG_TreatmentFOLFIRINOX.csv", header = TRUE)
dataset$X <- gsub('-mRNA', '', dataset$X)

# identifier mapping to Entrez Gene
hgcn2entrez <- clusterProfiler::bitr(dataset$X, fromType = "SYMBOL",toType = c("ENTREZID","SYMBOL"), OrgDb = org.Hs.eg.db)

'select()' returned 1:1 mapping between keys and columns
0.72% of input gene IDs are fail to map...

data.mapped <- merge(dataset, hgcn2entrez, by.x="X", by.y="SYMBOL", all.x = TRUE)

# filter genes without Entrez Gene identifier
data <- data.mapped %>% tidyr::drop_na(ENTREZID)

data.up <- unique(data[data$P.value < 0.05 & data$Log2.fold.change > 0.58, c(1,2)])
data.down <- unique(data[data$P.value < 0.05 & data$Log2.fold.change < -0.58, c(1,2)])

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PPI network analysis

Next, we will create a protein-protein interaction network with all differentially expressed genes using the STRING database.

commandsRun(paste0('string protein query cutoff=0.7 newNetName="PPI network" query="',query,'" limit=0'))

[1] "Loaded network 'STRING network - PPI network - 4' with 84 nodes and 192 edges"

Let’s explore the network

• Q1: How many of the differentially expressed genes were found in STRING?
• Q2: Are all genes connected in the network?
• Q3: Change the confidence cutoff in the commandsRun call from 0.7 (high confidence) to 0.4 (medium confidence). What changes?

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Data visualization

Use the same visualization you created in workflow 1 to visualize the gene expression data on the network.

loadTableData(data, data.key.column = "ENTREZID", table.key.column = "query term")

[1] "Success: Data loaded in defaultnode table"

RCy3::copyVisualStyle("default","ppi")
RCy3::setNodeLabelMapping("display name", style.name="ppi")

NULL

RCy3::lockNodeDimensions(TRUE, style.name="ppi")
data.values<-c(-1,0,1) 
node.colors <- c(rev(brewer.pal(length(data.values), "RdBu")))
setNodeColorMapping("Log2.fold.change", data.values, node.colors, default.color = "#99FF99", style.name = "ppi")

NULL

RCy3::setVisualStyle("ppi")

                message 
"Visual Style applied." 

RCy3::toggleGraphicsDetails()

Interpretation

• Q4: Do you see clusters of up- or down-regulated genes in the PPI network?

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Pathway information

Next, we will add information about participation of the differentially expressed genes in molecular pathway models.

# run CyTargetLinker

wp <- file.path(getwd(), "data/wikipathways-hsa-20200710.xgmml")

commandsRun(paste0('cytargetlinker extend idAttribute="query term" linkSetFiles="', wp, '"'))

[1] "Extension step: 1"                                                            
[2] "Linkset: WikiPathways pathway-gene network_Homo sapiens_WikiPathways_20200710"
[3] "Added edges: 612"                                                             
[4] "Added nodes: 258"                                                             

commandsRun('cytargetlinker applyLayout network="current"')
commandsRun('cytargetlinker applyVisualstyle network="current"')
RCy3::setNodeLabelMapping("display name", style.name="CyTargetLinker")

NULL

# there is an issue in the latest version with visualization of the added edges - the workaround below solves this for now
RCy3::cloneNetwork()

network 
  43781 

RCy3::setVisualStyle("default")

                message 
"Visual Style applied." 

RCy3::setVisualStyle("CyTargetLinker")

                message 
"Visual Style applied." 

# TODO: VISUAL STYLE
# adapt the visual style to also show the differential gene expression as the node fill color

Interpretation

• Q5: How many differentially expressed genes are in at least one of the pathways?
• Q6: Are the genes also functionally related based on the PPI network?

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Save Cytoscape output and session

# Saving output
png.file <- file.path(getwd(), "ppi-network.png")
exportImage(png.file,'PNG', zoom = 500)
cys.file <- file.path(getwd(), "ppi-network.cys")
saveSession(cys.file) 

#comment following line if you want to manipulate the visualization in Cytoscape
RCy3::closeSession(save.before.closing = F)