---
title: "SBGNview Based Pathway Analysis and Visualization Workflow"
author:
- Xiaoxi Dong
- Kovidh Vegesna, kvegesna (AT) uncc.edu
- Weijun Luo, luo_weijun (AT) yahoo.com
date: "`r format(Sys.time(), '%d %B, %Y')`"
output:
bookdown::html_document2:
fig_caption: yes
number_sections: yes
toc: yes
toc_float:
collapsed: false
editor_options:
chunk_output_type: console
bibliography: REFERENCES.bib
vignette: >
%\VignetteIndexEntry{Pathway analysis using SBGNview gene set}
\usepackage[utf8]{inputenc}
%\VignetteEngine{knitr::rmarkdown}
---
# Introduction
SBGNview has collected pathway data and gene sets from the following databases: Reactome, PANTHER Pathway, SMPDB, MetaCyc and MetaCrop. These gene sets can be used for pathway enrichment analysis.
In this vignette, we will show you a complete pathway analysis workflow based on GAGE + SBGNview. Similar workflows have been documented in the [gage package](https://bioconductor.org/packages/gage/) using GAGE + [Pathview](https://bioconductor.org/packages/pathview/).
# Citation
Please cite the following papers when using the open-source SBGNview package. This will help the project and our team:
Luo W, Brouwer C. Pathview: an R/Biocondutor package for pathway-based data integration and visualization. Bioinformatics, 2013, 29(14):1830-1831, <a href=https://doi.org/10.1093/bioinformatics/btt285>doi: 10.1093/bioinformatics/btt285</a>
Please also cite the GAGE paper when using the gage package:
Luo W, Friedman M, etc. GAGE: generally applicable gene set enrichment for pathway analysis. BMC Bioinformatics, 2009, 10, pp. 161, <a href=https://doi.org/10.1186/1471-2105-10-161>doi: 10.1186/1471-2105-10-161</a>
# Installation and quick start
Please see the [Quick Start tutorial](https://bioconductor.org/packages/devel/bioc/vignettes/SBGNview/inst/doc/SBGNview.quick.start.html) for installation instructions and quick start examples.
# Complete pathway analysis + visualization workflow
In this example, we analyze a RNA-Seq dataset of IFNg KO mice vs wild type mice. It contains normalized RNA-seq gene expression data described in Greer, Renee L., Xiaoxi Dong, et al, 2016.
## Load the gene (RNA-seq) data
The RNA abundance data was quantile normalized and log2 transformed, stored in a ["SummarizedExperiment"](https://bioconductor.org/packages/release/bioc/html/SummarizedExperiment.html) object. SBGNview input user data (gene.data or cpd.data) can be either a numeric matrix or a vector, like those in [pathview](https://bioconductor.org/packages/pathview/). In addition, it can be a "SummarizedExperiment" object, which is commonly used in BioConductor packages.
```{r , echo = TRUE, results = 'hide', message = FALSE, warning = FALSE}
library(SBGNview)
library(SummarizedExperiment)
data("IFNg", "pathways.info")
count.data <- assays(IFNg)$counts
head(count.data)
wt.cols <- which(IFNg$group == "wt")
ko.cols <- which(IFNg$group == "ko")
```
## Gene sets from SBGNview pathway collection
### Load gene set for mouse with ENSEMBL gene IDs
```{r , echo = TRUE , results = 'hide', message = FALSE, warning = FALSE}
ensembl.pathway <- sbgn.gsets(id.type = "ENSEMBL",
species = "mmu",
mol.type = "gene",
output.pathway.name = TRUE
)
head(ensembl.pathway[[2]])
```
### Pathway or gene set analysis using GAGE
```{r , echo = TRUE, results = 'hide', message = FALSE, warning = FALSE}
if(!requireNamespace("gage", quietly = TRUE)) {
BiocManager::install("gage", update = FALSE)
}
library(gage)
degs <- gage(exprs = count.data,
gsets = ensembl.pathway,
ref = wt.cols,
samp = ko.cols,
compare = "paired" #"as.group"
)
head(degs$greater)[,3:5]
head(degs$less)[,3:5]
down.pathways <- row.names(degs$less)[1:10]
head(down.pathways)
```
## Visualize perturbations in top SBGN pathways
### Calculate fold changes or gene perturbations
The abundance values were log2 transformed. Here we calculate the fold change of IFNg KO group v.s. WT group.
```{r , echo = TRUE, results = 'hide', message = FALSE, warning = FALSE}
ensembl.koVsWt <- count.data[,ko.cols]-count.data[,wt.cols]
head(ensembl.koVsWt)
#alternatively, we can also calculate mean fold changes per gene, which corresponds to gage analysis above with compare="as.group"
mean.wt <- apply(count.data[,wt.cols] ,1 ,"mean")
head(mean.wt)
mean.ko <- apply(count.data[,ko.cols],1,"mean")
head(mean.ko)
# The abundance values were on log scale. Hence fold change is their difference.
ensembl.koVsWt.m <- mean.ko - mean.wt
```
### Visualize pathway perturbations by SBNGview
```{r , echo = TRUE, results = 'hide', message = FALSE, warning = FALSE}
#load the SBGNview pathway collection, which may takes a few seconds.
data(sbgn.xmls)
down.pathways <- sapply(strsplit(down.pathways,"::"), "[", 1)
head(down.pathways)
sbgnview.obj <- SBGNview(
gene.data = ensembl.koVsWt,
gene.id.type = "ENSEMBL",
input.sbgn = down.pathways[1:2],#can be more than 2 pathways
output.file = "ifn.sbgnview.less",
show.pathway.name = TRUE,
max.gene.value = 2,
min.gene.value = -2,
mid.gene.value = 0,
node.sum = "mean",
output.format = c("png"),
font.size = 2.3,
org = "mmu",
text.length.factor.complex = 3,
if.scale.compartment.font.size = TRUE,
node.width.adjust.factor.compartment = 0.04
)
sbgnview.obj
```{r ifng, echo = FALSE,fig.cap="\\label{fig:ifng}SBGNview graph of the most down-regulated pathways in IFNg KO experiment"}
library(knitr)
include_graphics("ifn.sbgnview.less_R-HSA-877300_Interferon gamma signaling.svg")
```
```{r ifna, echo = FALSE,fig.cap="\\label{fig:ifna}SBGNview graph of the second most down-regulated pathways in IFNg KO experiment"}
library(knitr)
include_graphics("ifn.sbgnview.less_R-HSA-909733_Interferon alpha_beta signaling.svg")
```
## SBGNview with SummarizedExperiment object
The 'cancer.ds' is a microarray dataset from a breast cancer study. The dataset was adopted from gage package and processed into a SummarizedExperiment object. It is used to demo SBGNview's visualization ability.
```{r , echo = TRUE, results = 'hide', message = FALSE, warning = FALSE}
data("cancer.ds")
sbgnview.obj <- SBGNview(
gene.data = cancer.ds,
gene.id.type = "ENTREZID",
input.sbgn = "R-HSA-877300",
output.file = "demo.SummarizedExperiment",
show.pathway.name = TRUE,
max.gene.value = 1,
min.gene.value = -1,
mid.gene.value = 0,
node.sum = "mean",
output.format = c("png"),
font.size = 2.3,
org = "hsa",
text.length.factor.complex = 3,
if.scale.compartment.font.size = TRUE,
node.width.adjust.factor.compartment = 0.04
)
sbgnview.obj
```
```{r cancerds, echo = FALSE,fig.cap="\\label{fig:cancerds}SBGNview of a cancer dataset gse16873"}
include_graphics("demo.SummarizedExperiment_R-HSA-877300_Interferon gamma signaling.svg")
```
# Session Info
```{r}
sessionInfo()
```