--- title: "A workflow to study mechanistic indicators for driver gene prediction with Moonlight" author: "Mona Nourbakhsh^+^ , Astrid Saksager^+^, Nikola Tom, Xi Steven Chen, Antonio Colaprico, Catharina Olsen, Matteo Tiberti, Elena Papaleo" subtitle: ^+^ These authors contributed equally to the paper as first authors. date: "`r Sys.Date()`" output: BiocStyle::html_document: toc: true number_sections: false toc_depth: 2 highlight: haddock vignette: > %\VignetteIndexEntry{A workflow to study mechanistic indicators for driver gene prediction with Moonlight} %\VignetteEngine{knitr::rmarkdown} %\VignetteEncoding{UTF-8} references: - id: ref1 title: Orchestrating high-throughput genomic analysis with Bioconductor author: - family: Huber, Wolfgang and Carey, Vincent J and Gentleman, Robert and Anders, Simon and Carlson, Marc and Carvalho, Benilton S and Bravo, Hector Corrada and Davis, Sean and Gatto, Laurent and Girke, Thomas and others given: journal: Nature methods volume: 12 number: 2 pages: 115-121 issued: year: 2015 - id: ref2 title: GC-content normalization for RNA-Seq data author: - family: Risso, Davide and Schwartz, Katja and Sherlock, Gavin and Dudoit, Sandrine given: journal: BMC bioinformatics volume: 12 number: 1 pages: 480 issued: year: 2011 - id: ref3 title: Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments author: - family: Bullard, James H and Purdom, Elizabeth and Hansen, Kasper D and Dudoit, Sandrine given: journal: BMC bioinformatics volume: 11 number: 1 pages: 94 issued: year: 2010 - id: ref4 title: Inferring regulatory element landscapes and transcription factor networks from cancer methylomes author: - family: Yao, L., Shen, H., Laird, P. 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id: ref27 title: "A workflow to study mechanistic indicators for driver gene prediction with Moonlight" author: - family: Nourbakhsh, Mona and Saksager, Astrid and Tom, Nikola and Chen, Xi Steven and Colaprico, Antonio and Olsen, Catharina and Tiberti, Matteo and Papaleo, Elena given: journal: Briefings in Bioinformatics URL: "https://doi.org/10.1093/bib/bbad274" DOI: "10.1093/bib/bbad274" issued: year: 2023 - id: ref28 title: "EpiMix is an integrative tool for epigenomic subtyping using DNA methylation" author: - family: Zheng, Yuanning and Jun, John and Brennan, Kevin and Gevaert, Olivier given: journal: Cell Reports Methods URL: "https://doi.org/10.1016/j.crmeth.2023.100515" DOI: "10.1016/j.crmeth.2023.100515" issued: year: 2023 - id: ref29 title: "CScape-somatic: distinguishing driver and passenger point mutations in the cancer genome" author: - family: Rogers, Mark F and Gaunt, Tom R and Campbell, Colin given: journal: Bioinformatics URL: "https://doi.org/10.1093/bioinformatics/btaa242" DOI: "10.1093/bioinformatics/btaa242" issued: year: 2020 --- ```{r setup, include=FALSE} knitr::opts_chunk$set(dpi = 72) knitr::opts_chunk$set(cache=FALSE) ``` # Abstract In order to make light of cancer development, it is crucial to understand which genes play a role in the mechanisms linked to this disease and moreover which role that is. Commonly biological processes such as proliferation and apoptosis have been linked to cancer progression. We have developed the Moonlight framework that allows for prediction of cancer driver genes through multi-omics data integration. Based on expression data we perform functional enrichment analysis, infer gene regulatory networks and upstream regulator analysis to score the importance of well-known biological processes with respect to the studied cancer. We then use these scores to predict oncogenic mediators with two specific roles: genes that potentially act as tumor suppressor genes (TSGs) and genes that potentially act as oncogenes (OCGs). This constitutes Moonlight's primary layer. As gene expression data alone does not explain the cancer phenotypes, a second layer of evidence is needed. We have integrated two secondary layers, one based on mutations, and one based on abnormal DNA methylation patterns, for prediction of the driver genes. The mutational layer predicts driver mutations in the oncogenic mediators and thereby allows for the prediction of cancer driver genes using the driver mutation prediction tool CScape-somatic. The methylation layer investigates abnormal DNA methylation patterns and differentially methylated CpG sites in the oncogenic mediators using the tool EpiMix and uses patterns of hypo- and hypermethylation for prediction of the driver genes. Overall, this methodology not only allows us to identify genes with dual role (TSG in one cancer type and OCG in another) but also to elucidate the underlying biological processes. # Introduction Cancer development is influenced by (epi)genetic alterations in two distinctly different categories of genes, known as tumor suppressor genes (TSG) and oncogenes (OCG). The occurrence of these alterations in genes of the first category leads to faster cell proliferation while alterations in genes of second category increases or changes their function. In 2020, we developed the Moonlight framework that allows for prediction of cancer driver genes [@ref26]. Here, gene expression data are integrated together with biological processes and gene regulatory networks to score the importance of well-known biological processes with respect to the studied cancer. These scores are used to predict oncogenic mediators: putative TSGs and putative OCGs. As gene expression data alone is not enough to explain the deregulation of the genes, a second layer of evidence is needed. For this reason, we integrated a secondary mutational layer which predicts driver mutations and passenger mutations in the oncogenic mediators. The prediction of the driver mutations are carried out using the CScape-somatic [@ref29] driver mutation prediction tool. Those oncogenic mediators with at least one driver mutation are retained as the final set of driver genes [@ref27]. Moreover, we have integrated a secondary methylation layer that investigates abnormal DNA methylation patterns in the oncogenic mediators using the tool EpiMix [@ref28]. Those oncogenic mediators demonstrating hypo- and hypermethylation patterns are predicted as OCGs and TSGs, respectively. These new functionalities are released in the updated version of Moonlight to produce Moonlight2R. # Moonlight's pipeline Moonlight's pipeline is shown below: ```{r, fig.width=3, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("Moonlight2_pipeline_upd.png") ``` # Moonlight's proposed workflow The proposed pipeline consists of following eight steps: 1. The input to Moonlight is a set of differentially expressed genes between two biological conditions such as cancer and healthy samples or two cancer subtyoes. Besides differentially expressed genes, gene expression data is also needed. Mutation data and/or methylation data are also needed. 2. **FEA** Functional Enrichment Analysis, using Fisher's test, to identify gene sets (with biological functions linked to cancer) significantly enriched by regulated genes (RG). 3. **GRN** Gene Regulatory Network inferred between each single DEG (sDEG) and all genes by means of mutual information, obtaining for each DEG a list of RG. 4. **URA** Upstream Regulator Analysis for DEGs in each enriched gene set, we applied z-score being the ratio between the sum of all predicted effects for all the gene involved in the specific function and the square-root of the number of all genes. 5. **PRA** Pattern Recognition Analysis identifies putative TSGs (down) and OCGs (up). We either use user defined biological processes or random forests. 6. **DMA** Driver Mutation Analysis identifies driver mutations in the putative TSGs and OCGs through the driver mutation prediction tool, CScape-somatic. 7. **GMA** Gene Methylation Analysis identifies abnormal DNA methylation patterns in the putative TSGs and OCGs through the tool, EpiMix. 8. We apply the above procedure to multiple cancer types to obtain cancer-specific lists of TSGs and OCGs. We compare the lists for each cancer: if a sDEG is TSG in a cancer and OCG in another we define it as dual-role TSG-OCG. Otherwise if we find a sDEG defined as OCG or TSG only in one tissue we define it as tissue specific biomarker. 9. We use the COSMIC database to define a list of gold standard TSG and OCGs to assess the accuracy of the proposed method. # Installation To install Moonlight2R use the code below. ## Installation from BioConductor ```{r, eval = FALSE} if (!require("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("Moonlight2R") ``` ## Installation from GitHub First, install `devtools` or if you already have it installed, load it. ```{r, eval = FALSE} install.packages("devtools") library(devtools) ``` Install Moonlight2R from GitHub: ```{r, eval = FALSE} devtools::install_github(repo = "ELELAB/Moonlight2R") ``` ## Installation from GitHub with accompanying vignette First, install the BiocStyle Bioconductor package. ```{r, eval = FALSE} if (!require("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("BiocStyle") ``` Then install Moonlight2R with its accompanying vignette. ```{r, eval = FALSE} devtools::install_github(repo = "ELELAB/Moonlight2R", build_vignettes = TRUE) ``` You can view the vignette in the following way. ```{r, eval = FALSE} vignette( "Moonlight2R", package="Moonlight2R") ``` # Load libraries ```{r, eval = TRUE} library(Moonlight2R) library(magrittr) library(dplyr) ``` # `Obtain Input` The input to Moonlight is a set of differentially expressed genes and gene expression data. Mutation and/or methylation data are also needed. These data should be available for the same sample sets. Gene expression data, mutation data, methylation data and differentially expressed genes can for example be obtained from TCGA using the R package TCGAbiolinks. Help documents on how to use TCGAbiolinks are available [here](https://bioconductor.org/packages/release/bioc/vignettes/TCGAbiolinks/inst/doc/index.html). To find other examples of usage of TCGAbiolinks on TCGA cancer types see our [GitHub repository](https://github.com/ELELAB/LUAD_LUSC_TCGA_comparison). Example data of the input (differentially expressed genes, gene expression data, mutation data, and methylation data) are stored in the Moonlight2R package: ```{r, eval = TRUE} data(DEGsmatrix) data(dataFilt) data(dataMAF) data(GEO_TCGAtab) data(LOC_transcription) data(LOC_translation) data(LOC_protein) data(Oncogenic_mediators_mutation_summary) data(DEG_Mutations_Annotations) data(dataMethyl) data(DEG_Methylation_Annotations) data(Oncogenic_mediators_methylation_summary) data(MetEvidenceDriver) data(LUAD_sample_anno) ``` # `Download`: Get GEO data You can search GEO data using the `getDataGEO` function. GEO_TCGAtab: a 18x12 matrix that provides the GEO data set we matched to one of the 18 given TCGA cancer types ```{r, eval = TRUE, echo = TRUE} knitr::kable(GEO_TCGAtab, digits = 2, caption = "Table with GEO data set matched to one of the 18 given TCGA cancer types ", row.names = TRUE) ``` ## `getDataGEO`: Search by cancer type and data type [Gene Expression] The user can query and download the cancer types supported by GEO, using the function `getDataGEO`: ```{r, eval = TRUE, echo = TRUE, results='hide', warning = FALSE, message = FALSE} dataFilt_GEO <- getDataGEO(GEOobject = "GSE20347", platform = "GPL571") ``` ```{r, eval = TRUE, echo = TRUE, results='hide', warning = FALSE, message = FALSE} dataFilt_GEO <- getDataGEO(TCGAtumor = "ESCA") ``` ## `FEA`: Functional Enrichment Analysis The user can perform a functional enrichment analysis using the function `FEA`. For each DEG in the gene set a z-score is calculated. This score indicates how the genes act in the gene set. ```{r, eval = TRUE, echo = TRUE, results='hide'} data(DEGsmatrix) data(DiseaseList) data(EAGenes) dataFEA <- FEA(DEGsmatrix = DEGsmatrix) ``` The output can be visualized with a FEA plot. ## `FEAplot`: Functional Enrichment Analysis Plot The user can plot the result of a functional enrichment analysis using the function `plotFEA`. A negative z-score indicates that the process' activity is decreased. A positive z-score indicates that the process' activity is increased. ```{r, eval = TRUE, echo = TRUE, message=FALSE, results='hide', warning=FALSE} plotFEA(dataFEA = dataFEA, additionalFilename = "_exampleVignette", height = 10, width = 20) ``` The figure generated by the above code is shown below: ```{r, fig.width=3, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("FEAplot.gif") ``` ## `GRN`: Gene Regulatory Network The user can perform a gene regulatory network analysis using the function `GRN` which infers the network using the parmigene package. For illustrative purposes and to decrease runtime, we have set `nGenesPerm = 5` and `nBoot = 5` in the example below, however, we recommend setting these parameters to `nGenesPerm = 2000` and `nBoot = 400` to achieve optimal results, as they are set by default in the function arguments. ```{r, eval = TRUE} data(DEGsmatrix) data(dataFilt) dataGRN <- GRN(DEGsmatrix = DEGsmatrix, TFs = sample(rownames(DEGsmatrix), 100), normCounts = dataFilt, nGenesPerm = 5, kNearest = 3, nBoot = 5, DiffGenes = TRUE) ``` ## `URA`: Upstream Regulator Analysis The user can perform upstream regulator analysis using the function `URA`. This function is applied to each DEG in the enriched gene set and its neighbors in the GRN. ```{r, eval = TRUE, echo = TRUE, results='hide'} data(dataGRN) data(DEGsmatrix) data(DiseaseList) data(EAGenes) dataFEA <- FEA(DEGsmatrix = DEGsmatrix) BPselected <- dataFEA$Diseases.or.Functions.Annotation[1:5] dataURA <- URA(dataGRN = dataGRN, DEGsmatrix = DEGsmatrix, BPname = BPselected, nCores=1) ``` ## `PRA`: Pattern Regognition Analysis The user can retrieve putative TSG/OCG using either selected biological processes in the expert-based approach or a random forest classifier trained on known COSMIC OCGs/TSGs in the machine learning approach. The user must enter the chosen biological processes in the `BPname` argument to use the expert-based approach or set `BPname = NULL` to use the machine learning approach. ```{r, eval = TRUE} data(dataURA) data(tabGrowBlock) data(knownDriverGenes) dataPRA <- PRA(dataURA = dataURA, BPname = c("apoptosis","proliferation of cells"), thres.role = 0) ``` ## `DMA`: Driver Mutation Analysis The user can identify driver mutations with `DMA` in the oncogenic mediators established by `PRA`. The passenger or driver status is estimated with CScape-somatic [@ref29]. This function will further generate three files: DEG_Mutations_Annotations.rda, Oncogenic_mediators_mutation_summary.rda and cscape_somatic_output.rda. These will be placed in the specified results-folder. The user needs to download two CScape-somatic files in order to run DMA named css_coding.vcf.gz and css_noncoding.vcf.gz, respectively. These two files can be downloaded at http://cscape-somatic.biocompute.org.uk/#download. The corresponding .tbi files (css_coding.vcf.gz.tbi and css_noncoding.vcf.gz.tbi) must also be downloaded and be placed in the same folder. ```{r, eval = FALSE} data(dataPRA) data(dataMAF) data(DEGsmatrix) data(LOC_transcription) data(LOC_translation) data(LOC_protein) data(NCG) data(EncodePromoters) dataDMA <- DMA(dataMAF = dataMAF, dataDEGs = DEGsmatrix, dataPRA = dataPRA, results_folder = "DMAresults", coding_file = "css_coding.vcf.gz", noncoding_file = "css_noncoding.vcf.gz") ``` ## `GMA`: Gene Methylation Analysis The user can predict driver genes with `GMA` following prediction of the oncogenic mediators established by `PRA`. `GMA` predicts driver genes by investigating abnormal DNA methylation patterns in the oncogenic mediators using the tool EpiMix [@ref28]. Oncogenic mediators with hypo- and hypermethylation patterns are predicted as OCGs and TSGs, respectively. This function will generate these files: DEG_Methylation_Annotations.rda, Oncogenic_mediators_methylation_summary.rda, EpiMix_Results_Enhancer.rds, EpiMix_Results_Regular.rds, FunctionalPairs_Enhancer.csv, FunctionalPairs_Regular.csv, and FunctionalProbes_Regular.rds. These will be placed in the specified results-folder. ```{r, eval = TRUE} data("dataMethyl") data("dataFilt") data("dataPRA") data("DEGsmatrix") data("LUAD_sample_anno") data("NCG") data("EncodePromoters") data("MetEvidenceDriver") # Subset column names (sample names) in expression data to patient level pattern <- "^(.{4}-.{2}-.{4}-.{2}).*" colnames(dataFilt) <- sub(pattern, "\\1", colnames(dataFilt)) dataGMA <- GMA(dataMET = dataMethyl, dataEXP = dataFilt, dataPRA = dataPRA, dataDEGs = DEGsmatrix, sample_info = LUAD_sample_anno, met_platform = "HM450", prevalence_filter = NULL, output_dir = "./GMAresults", cores = 1, roadmap.epigenome.ids = "E096", roadmap.epigenome.groups = NULL) ``` ## `GLS`: Gene Literature Search The user can perform a literature search on driver genes using the `GLS` function. This function takes as input driver genes, a query and maximum number of records to retrieve from PubMed. Standard PubMed syntax can be used in the query. For example, Boolean operators AND, OR, NOT can be applied and tags such as [AU], [TITLE/ABSTRACT], [Affiliation] can be used. `GLS` fetches data of PubMed records matching the specified query and outputs PubMed IDs matching the query along with doi, title, abstract, year of publication, keywords, and total number of PubMed publications. This is done for each of the genes supplied in the input. ```{r, eval = TRUE} genes_query <- "BRCA1" dataGLS <- GLS(genes = genes_query, query_string = "AND cancer AND driver", max_records = 20) head(dataGLS) ``` ## `Level of consequence`: Effect of mutations on three different levels The user can investigate the predicted effect of different mutation types on the transcriptional level through the table LOC_transcription: ```{r} knitr::kable(LOC_transcription) ``` The user can investigate the predicted effect of different mutation types on the translational level through the table LOC_translation: ```{r} knitr::kable(LOC_translation) ``` The user can investigate the predicted effect of different mutation types on the protein level through the table LOC_protein: ```{r} knitr::kable(LOC_protein) ``` ## `plotNetworkHive`: GRN hive visualization taking into account COSMIC cancer genes In the following plot the nodes are separated into three groups: known tumor suppressor genes (yellow), known oncogenes (green) and the rest (gray). ```{r, eval = TRUE, echo = TRUE, results='hide', warning = FALSE, message = FALSE} data(knownDriverGenes) data(dataGRN) plotNetworkHive(dataGRN, knownDriverGenes, 0.55) ``` ## `plotDMA`: Heatmap of the driver/passenger status of mutations in TSGs/OCGs In the following plot the driver genes with driver mutations are shown. ```{r, eval = TRUE, warning = FALSE, message = FALSE, include=TRUE} data(dataDMA) data(DEG_Mutations_Annotations) data(Oncogenic_mediators_mutation_summary) plotDMA(DEG_Mutations_Annotations, Oncogenic_mediators_mutation_summary, type = 'complete', additionalFilename = "") ``` ```{r, fig.width=3, fig.height=4, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("heatmap_complete.gif") ``` ## `plotMoonlight`: Heatmap of Moonlight Gene Z-scores for mutation-driven TSGs/OCGs In the following plot the top 50 genes with the most driver mutations are visualised. The values are the moonlight gene z-score for the two biological processes ```{r, eval = TRUE, echo = TRUE, results='hide', warning = FALSE, message = FALSE} data(DEG_Mutations_Annotations) data(Oncogenic_mediators_mutation_summary) data(dataURA_plot) plotMoonlight(DEG_Mutations_Annotations, Oncogenic_mediators_mutation_summary, dataURA_plot, gene_type = "drivers", n = 50) ``` ```{r, fig.width=3, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("moonlight_heatmap.gif") ``` ## `plotGMA`: Heatmap of hypo/hyper/dual methylated CpG sites in TSGs/OCGs This function plots results of the `GMA`. It visualizes the number of hypo/hyper/dual methylated CpG sites in oncogenic mediators or in a user-supplied gene list. The results are visualized either in a single heatmap or split into different ones which is specified in the function's three modes: split, complete and genelist. ```{r, eval = TRUE, warning = FALSE, message = FALSE, include = TRUE} data("DEG_Methylation_Annotations") data("Oncogenic_mediators_methylation_summary") genes <- c("ACAN", "ACE2", "ADAM19", "AFAP1L1") plotGMA(DEG_Methylation_Annotations = DEG_Methylation_Annotations, Oncogenic_mediators_methylation_summary = Oncogenic_mediators_methylation_summary, type = "genelist", genelist = genes, additionalFilename = "./GMAresults/") ``` ```{r, fig.width=3, fig.height=4, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("heatmap_genelist_met.png") ``` ## `plotMoonlightMet`: Heatmap of Moonlight Gene Z-scores for methylation-driven TSGs/OCGs This function visualizes the effect of genes on biological processes and total number of hypo/hyper/dual methylated CpG sites in genes. ```{r, eval = TRUE, warning = FALSE, message = FALSE, include = TRUE} data("DEG_Methylation_Annotations") data("Oncogenic_mediators_methylation_summary") data("dataURA_plot") genes <- c("ACAN", "ACE2", "ADAM19", "AFAP1L1") plotMoonlightMet(DEG_Methylation_Annotations = DEG_Methylation_Annotations, Oncogenic_mediators_methylation_summary = Oncogenic_mediators_methylation_summary, dataURA = dataURA_plot, genes = genes, additionalFilename = "./GMAresults/") ``` ```{r, fig.width=3, fig.height=4, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("moonlight_heatmap_met.png") ``` ## `plotMetExp`: Visualize results from EpiMix of expression and methylation in genes This function visualizes results of EpiMix [@ref28] in three plots: one that visualizes the distribution of DNA methylation data (MixtureModelPlot), one that visualizes gene expression levels (ViolinPlot) and one that visualizes relationship between DNA methylation and gene expression (CorrelationPlot). ```{r, eval = TRUE, warning = FALSE, message = FALSE, include = TRUE} data("EpiMix_Results_Regular") data("dataMethyl") data("dataFilt") # Subset column names (sample names) in expression data to patient level pattern <- "^(.{4}-.{2}-.{4}-.{2}).*" colnames(dataFilt) <- sub(pattern, "\\1", colnames(dataFilt)) plotMetExp(EpiMix_results = EpiMix_Results_Regular, probe_name = "cg03035704", dataMET = dataMethyl, dataEXP = dataFilt, gene_of_interest = "ACVRL1", additionalFilename = "./GMAresults/") ``` ```{r, fig.width=3, fig.height=4, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("plotMetExp.png") ``` # Moonlight Analysis: Case Studies ### Introduction This vignette shows a complete workflow of the 'Moonlight2R' package. The code is divided into the following case studies: * 1. Predicting oncogenic mediators using Moonlight's primary layer * 2. Moonlight pipeline in one function * 3. Moonlight with driver mutation analysis * 4. Moonlight with gene methylation analysis ## Case study n. 1: Predicting oncogenic mediators using Moonlight's primary layer For illustrative purposes and to decrease runtime, we have set `nGenesPerm = 5` and `nBoot = 5` in the call of `GRN` in the following code block, however, we recommend setting these parameters to `nGenesPerm = 2000` and `nBoot = 400` to achieve optimal results, as they are set by default in the function arguments. ```{r,eval = TRUE,echo=TRUE,message=FALSE,warning=FALSE, results='hide'} data(DEGsmatrix) data(dataFilt) data(DiseaseList) data(EAGenes) data(tabGrowBlock) data(knownDriverGenes) dataFEA <- FEA(DEGsmatrix = DEGsmatrix) dataGRN <- GRN(TFs = sample(rownames(DEGsmatrix), 100), DEGsmatrix = DEGsmatrix, DiffGenes = TRUE, normCounts = dataFilt, nGenesPerm = 5, nBoot = 5, kNearest = 3) dataURA <- URA(dataGRN = dataGRN, DEGsmatrix = DEGsmatrix, BPname = c("apoptosis", "proliferation of cells")) dataDual <- PRA(dataURA = dataURA, BPname = c("apoptosis", "proliferation of cells"), thres.role = 0) oncogenic_mediators <- list("TSG"=names(dataDual$TSG), "OCG"=names(dataDual$OCG)) ``` ## `plotURA`: Upstream regulatory analysis plot The user can plot the result of the upstream regulatory analysis using the function `plotURA`. ```{r, eval = TRUE,message=FALSE,warning=FALSE, results='hide'} data(dataURA) plotURA(dataURA = dataURA, additionalFilename = "_exampleVignette") ``` The figure resulted from the code above is shown below: ```{r, fig.width=3, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("URAplot.gif") ``` ## Case study n. 2: Moonlight pipeline in one function For illustrative purposes and to decrease runtime, we have set `nGenesPerm = 5` and `nBoot = 5` in the following code block, however, we recommend setting these parameters to `nGenesPerm = 2000` and `nBoot = 400` to achieve optimal results, as they are set by default in the function arguments. ```{r,eval = FALSE,echo=TRUE,message=FALSE,warning=FALSE} data(dataFilt) data(DEGsmatrix) data(dataMAF) data(DiseaseList) data(EAGenes) data(tabGrowBlock) data(knownDriverGenes) data(LOC_transcription) data(LOC_translation) data(LOC_protein) data(NCG) data(EncodePromoters) listMoonlight <- moonlight(dataDEGs = DEGsmatrix, dataFilt = dataFilt, nTF = 100, DiffGenes = TRUE, nGenesPerm = 5, nBoot = 5, BPname = c("apoptosis","proliferation of cells"), dataMAF = dataMAF, path_cscape_coding = "css_coding.vcf.gz", path_cscape_noncoding = "css_noncoding.vcf.gz") save(listMoonlight, file = paste0("listMoonlight_ncancer4.Rdata")) ``` ## `plotCircos`: Moonlight Circos Plot An example of running Moonlight on five cancer types is visualized below in a circos plot. Outer ring: color by cancer type, Inner ring: OCGs and TSGs, Inner connections: green: common OCGs yellow: common TSGs red: possible dual role ```{r, eval = TRUE, echo = TRUE, results='hide', warning = FALSE, message = FALSE} data(listMoonlight) plotCircos(listMoonlight = listMoonlight, additionalFilename = "_ncancer5") ``` The figure generated by the code above is shown below: ```{r, fig.width=3, echo = FALSE, fig.align="center",hide=TRUE, message=FALSE,warning=FALSE} knitr::include_graphics("circos_ocg_tsg_ncancer5.gif") ``` ## Case study n. 3: Moonlight with driver mutation analysis For illustrative purposes and to decrease runtime, we have set `nGenesPerm = 5` and `nBoot = 5` in the call of `GRN` in the following code block, however, we recommend setting these parameters to `nGenesPerm = 2000` and `nBoot = 400` to achieve optimal results, as they are set by default in the function arguments. ```{r,eval = FALSE,echo=TRUE,message=FALSE,warning=FALSE} data(DEGsmatrix) data(dataFilt) data(dataMAF) data(DiseaseList) data(EAGenes) data(tabGrowBlock) data(knownDriverGenes) data(LOC_transcription) data(LOC_translation) data(LOC_protein) data(NCG) data(EncodePromoters) # Perform gene regulatory network analysis dataGRN <- GRN(TFs = rownames(DEGsmatrix), DEGsmatrix = DEGsmatrix, DiffGenes = TRUE, normCounts = dataFilt, nGenesPerm = 5, kNearest = 3, nBoot = 5) # Perform upstream regulatory analysis # As example, we use apoptosis and proliferation of cells as the biological processes dataURA <- URA(dataGRN = dataGRN, DEGsmatrix = DEGsmatrix, BPname = c("apoptosis", "proliferation of cells"), nCores = 1) # Perform pattern recognition analysis dataPRA <- PRA(dataURA = dataURA, BPname = c("apoptosis", "proliferation of cells"), thres.role = 0) # Perform driver mutation analysis dataDMA <- DMA(dataMAF = dataMAF, dataDEGs = DEGsmatrix, dataPRA = dataPRA, results_folder = "DMAresults", coding_file = "css_coding.vcf.gz", noncoding_file = "css_noncoding.vcf.gz") ``` Next, we analyze the predicted driver genes and their mutations. ```{r, eval = TRUE} data(Oncogenic_mediators_mutation_summary) data(DEG_Mutations_Annotations) # Extract oncogenic mediators that contain at least one driver mutation # These are the driver genes knitr::kable(Oncogenic_mediators_mutation_summary %>% filter(!is.na(CScape_Driver))) # Extract mutation annotations of the predicted driver genes driver_mut <- DEG_Mutations_Annotations %>% filter(!is.na(Moonlight_Oncogenic_Mediator), CScape_Mut_Class == "Driver") # Extract driver genes with a predicted effect on the transcriptional level transcription_mut <- Oncogenic_mediators_mutation_summary %>% filter(!is.na(CScape_Driver)) %>% filter(Transcription_mut_sum > 0) # Extract mutation annotations of predicted driver genes that have a driver mutation # in its promoter region with a potential effect on the transcriptional level promoters <- DEG_Mutations_Annotations %>% filter(!is.na(Moonlight_Oncogenic_Mediator), CScape_Mut_Class == "Driver", Potential_Effect_on_Transcription == 1, Annotation == 'Promoter') ``` ## Case study n. 4: Moonlight with gene methylation analysis For illustrative purposes and to decrease runtime, we have set `nGenesPerm = 5` and `nBoot = 5` in the call of `GRN` in the following code block, however, we recommend setting these parameters to `nGenesPerm = 2000` and `nBoot = 400` to achieve optimal results, as they are set by default in the function arguments. ```{r,eval = TRUE,echo=TRUE,message=FALSE,warning=FALSE,results='hide'} data(DEGsmatrix) data(dataFilt) data(dataMAF) data(DiseaseList) data(EAGenes) data(tabGrowBlock) data(knownDriverGenes) data(LOC_transcription) data(LOC_translation) data(LOC_protein) data(NCG) data(EncodePromoters) data(dataMethyl) data(LUAD_sample_anno) data(MetEvidenceDriver) # Perform gene regulatory network analysis dataGRN <- GRN(TFs = rownames(DEGsmatrix), DEGsmatrix = DEGsmatrix, DiffGenes = TRUE, normCounts = dataFilt, nGenesPerm = 5, kNearest = 3, nBoot = 5) # Perform upstream regulatory analysis # As example, we use apoptosis and proliferation of cells as the biological processes dataURA <- URA(dataGRN = dataGRN, DEGsmatrix = DEGsmatrix, BPname = c("apoptosis", "proliferation of cells"), nCores = 1) # Perform pattern recognition analysis dataPRA <- PRA(dataURA = dataURA, BPname = c("apoptosis", "proliferation of cells"), thres.role = 0) # Subset column names (sample names) in expression data to patient level pattern <- "^(.{4}-.{2}-.{4}-.{2}).*" colnames(dataFilt) <- sub(pattern, "\\1", colnames(dataFilt)) # Perform gene methylation analysis dataGMA <- GMA(dataMET = dataMethyl, dataEXP = dataFilt, dataPRA = dataPRA, dataDEGs = DEGsmatrix, sample_info = LUAD_sample_anno, met_platform = "HM450", prevalence_filter = NULL, output_dir = "./GMAresults", cores = 1, roadmap.epigenome.ids = "E096", roadmap.epigenome.groups = NULL) ``` ## Citation Please cite the MoonlightR and Moonlight2R packages: * "Interpreting pathways to discover cancer driver genes with Moonlight." Nature Communications (2020): [10.1038/s41467-019-13803-0](https://doi.org/10.1038/s41467-019-13803-0). [@ref26] * "A workflow to study mechanistic indicators for driver gene prediction with Moonlight." Briefings in Bioinformatics (2023): [10.1093/bib/bbad274](https://doi.org/10.1093/bib/bbad274). [@ref27] Related publications to this vignette: * "TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data." Nucleic acids research (2015): [gkv1507](http://dx.doi.org/doi:10.1093/nar/gkv1507). [@ref25] * "TCGA Workflow: Analyze cancer genomics and epigenomics data using Bioconductor packages". F1000Research [10.12688/f1000research.8923.1](http://dx.doi.org/doi:10.12688/f1000research.8923.1) [@ref24] ****** Session Information ****** ```{r sessionInfo} sessionInfo() ``` # References