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README.md
<!-- badges: start --> [![R-CMD-check](https://github.com/pouryany/PanomiR/workflows/R-CMD-check/badge.svg)](https://github.com/pouryany/PanomiR/actions) [![lint](https://github.com/pouryany/PanomiR/workflows/lint/badge.svg)](https://github.com/pouryany/PanomiR/actions) <!-- badges: end --> ## Introduction PanomiR is a package for pathway and microRNA Analysis of gene expression data. This document provides details about how to install and utilize various functionality in PanomiR. For questions, comments, and other queries, contact <pouryany@gmail.com> ## Installation PanomiR can be accessed via Bioconductor. To install, start R (version \> “4.2”) and run the following code. ``` r if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("PanomiR") ``` You can also install the latest development version of PanomiR using GitHub. ``` r devtools::install_github("pouryany/PanomiR") ``` ## 1. Pathway summarization PanomiR can generate pathway activity profiles given a gene expression dataset and a list of pathways. This section uses an example dataset from the Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) dataset to generate Pathway summary statistics. ``` r library(PanomiR) # Downloading example LIHC expression dataset LIHC_url <- url(paste0("https://github.com/pouryany/PanomiR_paper", "/raw/main/data/TCGA_LIHC.RDS")) TCGA_LIHC <- readRDS(LIHC_url) # Downloading example LIHC covariates dataset LIHC_cov_url <- url(paste0("https://github.com/pouryany/PanomiR_paper", "/raw/main/data/covariates_TCGA_LIHC.RDS")) cov_TCGA_LIHC <- readRDS(LIHC_cov_url) # Pathway reference from the PanomiR package data("path_gene_table") # Generating pathway summary statistics summaries <- pathwaySummary(TCGA_LIHC,path_gene_table,method = "x2") head(summaries)[,1:2] ``` ## TCGA-2V-A95S-01A-11R-A37K-07 ## Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS 107100510 ## Pathway.KEGG_CITRATE_CYCLE_TCA_CYCLE 137540732 ## Pathway.KEGG_PENTOSE_PHOSPHATE_PATHWAY 105321837 ## Pathway.KEGG_PENTOSE_AND_GLUCURONATE_INTERCONVERSIONS 115609938 ## Pathway.KEGG_FRUCTOSE_AND_MANNOSE_METABOLISM 78860500 ## Pathway.KEGG_GALACTOSE_METABOLISM 88480592 ## TCGA-2Y-A9GS-01A-12R-A38B-07 ## Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS 131945603 ## Pathway.KEGG_CITRATE_CYCLE_TCA_CYCLE 149435696 ## Pathway.KEGG_PENTOSE_PHOSPHATE_PATHWAY 135163359 ## Pathway.KEGG_PENTOSE_AND_GLUCURONATE_INTERCONVERSIONS 162386997 ## Pathway.KEGG_FRUCTOSE_AND_MANNOSE_METABOLISM 92557981 ## Pathway.KEGG_GALACTOSE_METABOLISM 103639806 ## 2. Differential Pathway activation Once you generate the pathway activity profiles, as discussed in the last section, there are several analysis that you can perform. We have bundled some of the most important ones into standalone functions. Here, we describe differential pathway activation profiling, which is examining differenes in pathway activity profiles in user-determined conditions. ``` r output0 <- differentialPathwayAnalysis(geneCounts = TCGA_LIHC, pathways = path_gene_table, covariates = cov_TCGA_LIHC, condition = 'shortLetterCode', adjustCovars ='plate') de.paths <- output0$DEP head(de.paths) ``` ## logFC AveExpr ## Pathway.REACTOME_NUCLEAR_SIGNALING_BY_ERBB4 -0.4006321 -0.708800 ## Pathway.KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION -0.4031298 -2.381353 ## Pathway.KEGG_JAK_STAT_SIGNALING_PATHWAY -0.3663317 -1.076826 ## Pathway.REACTOME_CLASS_A1_RHODOPSIN_LIKE_RECEPTORS -0.4057902 -2.098614 ## Pathway.REACTOME_GPCR_LIGAND_BINDING -0.3692202 -1.998562 ## Pathway.REACTOME_HDL_MEDIATED_LIPID_TRANSPORT -0.9826705 1.460851 ## t P.Value ## Pathway.REACTOME_NUCLEAR_SIGNALING_BY_ERBB4 -13.26122 1.633566e-33 ## Pathway.KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION -13.24757 1.853574e-33 ## Pathway.KEGG_JAK_STAT_SIGNALING_PATHWAY -12.58752 7.847204e-31 ## Pathway.REACTOME_CLASS_A1_RHODOPSIN_LIKE_RECEPTORS -12.30811 9.768330e-30 ## Pathway.REACTOME_GPCR_LIGAND_BINDING -12.24499 1.720745e-29 ## Pathway.REACTOME_HDL_MEDIATED_LIPID_TRANSPORT -11.89976 3.721198e-28 ## adj.P.Val B ## Pathway.REACTOME_NUCLEAR_SIGNALING_BY_ERBB4 1.130680e-30 65.36049 ## Pathway.KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION 1.130680e-30 65.23564 ## Pathway.KEGG_JAK_STAT_SIGNALING_PATHWAY 3.191196e-28 59.26021 ## Pathway.REACTOME_CLASS_A1_RHODOPSIN_LIKE_RECEPTORS 2.979341e-27 56.76963 ## Pathway.REACTOME_GPCR_LIGAND_BINDING 4.198618e-27 56.21046 ## Pathway.REACTOME_HDL_MEDIATED_LIPID_TRANSPORT 7.566436e-26 53.17513 ## contrast ## Pathway.REACTOME_NUCLEAR_SIGNALING_BY_ERBB4 shortLetterCodeTP-shortLetterCodeNT ## Pathway.KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION shortLetterCodeTP-shortLetterCodeNT ## Pathway.KEGG_JAK_STAT_SIGNALING_PATHWAY shortLetterCodeTP-shortLetterCodeNT ## Pathway.REACTOME_CLASS_A1_RHODOPSIN_LIKE_RECEPTORS shortLetterCodeTP-shortLetterCodeNT ## Pathway.REACTOME_GPCR_LIGAND_BINDING shortLetterCodeTP-shortLetterCodeNT ## Pathway.REACTOME_HDL_MEDIATED_LIPID_TRANSPORT shortLetterCodeTP-shortLetterCodeNT ## 3. Finding clusters of pathways PanomiR provides a function to find groups coordinated differentially activated pathways based on the PCxN methodology. ``` r # using an updated version of pcxn pcxn_url <- url(paste0("https://github.com/pouryany/PanomiR_paper", "/raw/main/data/pcxn_panomir.RDS")) pcxn_net <- readRDS(pcxn_url) set.seed(2) pathwayClustsLIHC <- mappingPathwaysClusters(pcxn = pcxn_net, dePathways = de.paths[1:300,], topPathways = 200, outDir=".", plotOut = FALSE, subplot = FALSE, prefix='', clusteringFunction = "cluster_louvain", correlationCutOff = 0.1) head(pathwayClustsLIHC$Clustering) ``` ## Pathway cluster ## 1 Pathway.KEGG_TRYPTOPHAN_METABOLISM 6 ## 2 Pathway.KEGG_BETA_ALANINE_METABOLISM 3 ## 3 Pathway.KEGG_LINOLEIC_ACID_METABOLISM 3 ## 4 Pathway.KEGG_RETINOL_METABOLISM 3 ## 5 Pathway.KEGG_DRUG_METABOLISM_CYTOCHROME_P450 3 ## 6 Pathway.KEGG_DNA_REPLICATION 2 ## 4. Prioritizing miRNAs per cluster of pathways. PanomiR identifies miRNAs that target clusters of pathways, as defined in the last section. In order to this, you would need a reference table of miRNA-Pathway association score (enrichment). We recommend using a customized miRNA-Pathway association table, tailored to your experimental data. Here, we provide a pre-processed table for LIHC table and in the next section, we will explain how to generate the customized tables. Note that in the example below, we use a sampling rate of 50, the recommended rate is between 500-1000. Also, we set the saveSampling argument to FALSE. This argument should be set to TRUE if you wish to save your sampling and check for different outputs from the clustering algorithms or pathway thresholds. ``` r # using an updated version of pcxn enrch_url <- url(paste0("https://github.com/pouryany/PanomiR_paper", "/raw/main/data/LIHC_ENRICHMENT_TargetScan03.RDS")) tableEnrich <- readRDS(enrch_url) set.seed(1) output2 <- prioritizeMicroRNA(enriches0 = tableEnrich, pathClust = pathwayClustsLIHC$Clustering, topClust = 1, sampRate = 50, method = c("aggInv"), outDir = "Output/", dataDir = "outData/", saveSampling = FALSE, runJackKnife = FALSE, numCores = 1, prefix = "outmiR", saveCSV = FALSE) ``` ## [1] "Working on Cluster1." ## [1] "Performing aggInv function." ## [1] "aggInv Method Done" ``` r head(output2$Cluster1) ``` ## x cluster_hits aggInv_cover aggInv_pval aggInv_fdr ## 1 hsa-miR-371a-5p 0 -0.98581204 2.166514e-46 9.402670e-44 ## 2 hsa-miR-505-3p.2 0 -1.16947734 6.777526e-31 1.470723e-28 ## 3 hsa-miR-556-5p 0 -0.89339753 6.940239e-28 1.004021e-25 ## 4 hsa-miR-1298-5p 0 -1.94996850 8.025049e-27 8.707179e-25 ## 5 hsa-miR-1278 1 -0.40782882 2.378328e-26 2.064388e-24 ## 6 hsa-miR-325-3p 3 -0.08758886 2.755195e-25 1.992925e-23 ## 5. miRNA-Pathway enrichment tables PanomiR best performs on tissue/experiment-customized datasets. In order to do this, you need to create a customized enrichment table. You can simply do so by using the pathway and miRNA list that we have provided as a part of the package. simply, plug in the name of the genes present (expressed) in your experiment in the following code ``` r # using an updated version of pcxn data("msigdb_c2") data("targetScan_03") customeTableEnrich <- miRNAPathwayEnrichment(mirSets = targetScan_03, pathwaySets = msigdb_c2, geneSelection = yourGenes, mirSelection = yourMicroRNAs, fromID = "ENSEMBL", toID = "ENTREZID", minPathSize = 9, numCores = 1, outDir = ".", saveOutName = NULL) ``` In the above section, the field `fromID` denotes the gene representation format of your input list. Here is a quick example that runs fast. ``` r # using an updated version of pcxn data("msigdb_c2") data("targetScan_03") tempEnrich <-miRNAPathwayEnrichment(targetScan_03[1:20],msigdb_c2[1:20]) head(tempEnrich) ``` ## x y ## 1 hsa-miR-101-3p.1 Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS ## 2 hsa-miR-1193 Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS ## 3 hsa-miR-1197 Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS ## 4 hsa-miR-124-3p.1 Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS ## 5 hsa-miR-124-3p.2/hsa-miR-506-3p Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS ## 6 hsa-miR-1252-5p Pathway.KEGG_GLYCOLYSIS_GLUCONEOGENESIS ## pval Intersect mirset_Size not_mirset pathway_Size ratio_in ratio_out ## 1 0.6041595 1 10 695 62 0.01639344 0.01294964 ## 2 0.3528596 2 14 691 62 0.03333333 0.01736614 ## 3 1.0000000 0 15 690 62 0.00000000 0.02173913 ## 4 0.4540258 3 28 677 62 0.05084746 0.03692762 ## 5 0.8722208 1 22 683 62 0.01639344 0.03074671 ## 6 1.0000000 0 16 689 62 0.00000000 0.02322206 ## ratio_ratios ## 1 1.2659381 ## 2 1.9194444 ## 3 0.0000000 ## 4 1.3769492 ## 5 0.5331772 ## 6 0.0000000 ## Session info ``` r sessionInfo() ``` ## R Under development (unstable) (2021-12-03 r81290) ## Platform: x86_64-apple-darwin17.0 (64-bit) ## Running under: macOS Mojave 10.14.6 ## ## Matrix products: default ## BLAS: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRblas.0.dylib ## LAPACK: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRlapack.dylib ## ## locale: ## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 ## ## attached base packages: ## [1] stats graphics grDevices utils datasets methods base ## ## other attached packages: ## [1] PanomiR_0.99.0 ## ## loaded via a namespace (and not attached): ## [1] igraph_1.2.9 knitr_1.36 magrittr_2.0.1 tidyselect_1.1.1 ## [5] R6_2.5.1 rlang_0.4.12 fastmap_1.1.0 fansi_0.5.0 ## [9] stringr_1.4.0 dplyr_1.0.7 tools_4.2.0 parallel_4.2.0 ## [13] xfun_0.28 utf8_1.2.2 withr_2.4.3 htmltools_0.5.2 ## [17] ellipsis_0.3.2 yaml_2.2.1 digest_0.6.29 tibble_3.1.6 ## [21] lifecycle_1.0.1 crayon_1.4.2 purrr_0.3.4 vctrs_0.3.8 ## [25] glue_1.5.1 evaluate_0.14 rmarkdown_2.11 limma_3.51.1 ## [29] stringi_1.7.6 compiler_4.2.0 pillar_1.6.4 forcats_0.5.1 ## [33] generics_0.1.1 pkgconfig_2.0.3