--- title: "An R package for Reactome Pathway Analysis" author: "\\ Guangchuang Yu ()\\ School of Public Health, The University of Hong Kong" date: "`r Sys.Date()`" bibliography: ReactomePA.bib csl: nature.csl output: BiocStyle::html_document: toc: true BiocStyle::pdf_document: toc: true vignette: > %\VignetteIndexEntry{An R package for Reactome Pathway Analysis} %\VignetteEngine{knitr::rmarkdown} %\VignetteDepends{org.Hs.eg.db} %\usepackage[utf8]{inputenc} --- ```{r style, echo=FALSE, results="asis", message=FALSE} BiocStyle::markdown() knitr::opts_chunk$set(tidy = FALSE, warning = FALSE, message = FALSE) ``` ```{r echo=FALSE, results='hide', message=FALSE} library(org.Hs.eg.db) library(DOSE) library(ReactomePA) ``` # Introduction This package is designed for reactome pathway-based analysis. Reactome is an open-source, open access, manually curated and peer-reviewed pathway database. # Citation If you use `r Biocpkg("Reactome")`[@yu_reactomepa_2016] in published research, please cite: __*G Yu*__, QY He^\*^. ReactomePA: an R/Bioconductor package for reactome pathway analysis and visualization. __*Molecular BioSystems*__ 2016, 12(2):477-479. doi:[10.1039/C5MB00663E](http://dx.doi.org/10.1039/C5MB00663E) # Supported organisms Currently `r Biocpkg("ReactomePA")` supports several model organisms, including 'celegans', 'fly', 'human', 'mouse', 'rat', 'yeast' and 'zebrafish'. The input gene ID should be Entrez gene ID. We recommend using `clusterProfiler::bitr` to convert biological IDs. For more detail, please refer to [bitr: Biological Id TranslatoR](http://www.bioconductor.org/packages/release/bioc/vignettes/clusterProfiler/inst/doc/clusterProfiler.html#bitr-biological-id-translator). # Pathway Enrichment Analysis Enrichment analysis is a widely used approach to identify biological themes. Here, we implement hypergeometric model to assess whether the number of selected genes associated with reactome pathway is larger than expected. The _p_ values were calculated based the hypergeometric model[@boyle2004]. ```{r} library(ReactomePA) data(geneList) de <- names(geneList)[abs(geneList) > 1.5] head(de) x <- enrichPathway(gene=de,pvalueCutoff=0.05, readable=T) head(as.data.frame(x)) ``` For calculation/parameter details, please refer to the vignette of `r Biocpkg("DOSE")`[@yu_dose_2015].. ## Pathway analysis of NGS data Pathway analysis using NGS data (eg, RNA-Seq and ChIP-Seq) can be performed by linking coding and non-coding regions to coding genes via `r Biocpkg("ChIPseeker")` package, which can annotates genomic regions to their nearest genes, host genes, and flanking genes respectivly. In addtion, it provides a function, __*seq2gene*__, that simultaneously considering host genes, promoter region and flanking gene from intergenic region that may under control via cis-regulation. This function maps genomic regions to genes in a many-to-many manner and facilitate functional analysis. For more details, please refer to `r Biocpkg("ChIPseeker")`[@yu_chipseeker_2015]. ## Visualize enrichment result We implement barplot, dotplot enrichment map and category-gene-network for visualization. It is very common to visualize the enrichment result in bar or pie chart. We believe the pie chart is misleading and only provide bar chart. ```{r fig.height=4, fig.width=7} barplot(x, showCategory=8) ``` ```{r fig.height=6, fig.width=8} dotplot(x, showCategory=15) ``` Enrichment map can be viusalized by __*enrichMap*__: ```{r fig.height=16, fig.width=20, eval=FALSE} enrichMap(x, layout=igraph::layout.kamada.kawai, vertex.label.cex = 1) ``` ![](figures/enrichMap.png) In order to consider the potentially biological complexities in which a gene may belong to multiple annotation categories, we developed __*cnetplot*__ function to extract the complex association between genes and diseases. ```{r fig.height=12, fig.width=12, eval=FALSE} cnetplot(x, categorySize="pvalue", foldChange=geneList) ``` ![](figures/cnetplot.png) ## Comparing enriched reactome pathways among gene clusters with clusterProfiler We have developed an `R` package `r Biocpkg("clusterProfiler")`[@yu_clusterprofiler:_2012] for comparing biological themes among gene clusters. `r Biocpkg("ReactomePA")` works fine with `r Biocpkg("clusterProfiler")` and can compare biological themes at reactome pathway perspective. ```{r fig.height=8, fig.width=13, eval=FALSE} require(clusterProfiler) data(gcSample) res <- compareCluster(gcSample, fun="enrichPathway") plot(res) ``` ![](figures/clusterProfiler.png) # Gene Set Enrichment Analysis A common approach in analyzing gene expression profiles was identifying differential expressed genes that are deemed interesting. The __*enrichPathway*__ function we demonstrated previously were based on these differential expressed genes. This approach will find genes where the difference is large, but it will not detect a situation where the difference is small, but evidenced in coordinated way in a set of related genes. Gene Set Enrichment Analysis (GSEA)[@subramanian_gene_2005] directly addressed this limitation. All genes can be used in GSEA; GSEA aggregates the per gene statistics across genes within a gene set, therefore making it possible to detect situations where all genes in a predefined set change in a small but coordinated way. For algorithm details, please refer to the vignette of `r Biocpkg("DOSE")`[@yu_dose_2015]. ```{r} y <- gsePathway(geneList, nPerm=1000, minGSSize=120, pvalueCutoff=0.2, pAdjustMethod="BH", verbose=FALSE) res <- as.data.frame(y) head(res) ``` ## Visualize GSEA result ```{r fig.height=16, fig.width=16, eval=FALSE} enrichMap(y) ``` ![](figures/enrichMap_gsea.png) ```{r fig.height=7, fig.width=10} gseaplot(y, geneSetID = "1280215") ``` # Pathway Visualization In `r Biocpkg("ReactomePA")`, we also implemented __*viewPathway*__ to visualized the pathway. ```{r fig.height=16, fig.width=16, eval=FALSE} viewPathway("E2F mediated regulation of DNA replication", readable=TRUE, foldChange=geneList) ``` ![](figures/E2F.png) More documents can be found on the project website, . # Session Information Here is the output of `sessionInfo()` on the system on which this document was compiled: ```{r echo=FALSE} sessionInfo() ``` # References