Last updated: 2024-06-25

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Knit directory: PPP/

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Unstaged changes:
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    Modified:   analysis/Identify_Kinase_Regulators.Rmd
    Modified:   analysis/KSEA.Rmd
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    Modified:   output/cnv/dset_hnsc.RDS
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Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.


These are the previous versions of the repository in which changes were made to the R Markdown (analysis/Differentially_Methylation.Rmd) and HTML (docs/Differentially_Methylation.html) files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view the files as they were in that past version.

File Version Author Date Message
Rmd 85fc7b1 Zhen Zuo 2024-06-24 .
html 85fc7b1 Zhen Zuo 2024-06-24 .

Load packages

library(viper)
library(aracne.networks)
library(dplyr)
library(plyr)
library(stringr)
library(Biobase)
library(EnsDb.Hsapiens.v86)
dir.create("output/DME/",showWarnings = FALSE)
dir.create("output/methy/",showWarnings = FALSE)

Define functions

read_exp <- function(file_name) {
    expr <- read.table(file_name, header = TRUE, sep = "\t", row.names = 1,
        as.is = TRUE)
    expr[is.na(expr)] <- 0
    n_0 <- count_zeros_in_rows(as.matrix(expr))
    # Remove features with more 20% zero/missing values
    expr <- expr[n_0 >= ncol(expr)/5, ]
    # Split rownames by |
    meta <- data.frame(rownames(expr))
    colnames(meta) <- c("ENSG")
    meta$ENSG <- sub("\\..*$", "", meta$ENSG)

    geneID <- ensembldb::select(EnsDb.Hsapiens.v86, keys = meta$ENSG, keytype = "GENEID",
        columns = c("SYMBOL", "UNIPROTID", "GENEID"))
    meta$UNIPROTID <- plyr::mapvalues(meta$ENSG, from = geneID$GENEID,
        to = geneID$UNIPROTID, warn_missing = FALSE)
    meta$SYMBOL <- plyr::mapvalues(meta$ENSG, from = geneID$GENEID, to = geneID$SYMBOL,
        warn_missing = FALSE)

    # Remove duplicated indexs
    binary_unique_index <- (!duplicated(meta$SYMBOL)) & (!is.na(meta$SYMBOL) &
        (!sapply(meta$SYMBOL, function(x) startsWith(x, "ENSG"))))

    print(table(binary_unique_index))

    meta <- meta[binary_unique_index, ]

    expr <- expr[binary_unique_index, ]
    rownames(expr) <- meta$SYMBOL

    rownames(meta) <- rownames(expr)
    return(list(expr, meta))
}

calculate_log_fold_change_and_pvalue <- function(data_matrix, group, adjust_method = "BH") {
    # Check if the length of the group variable matches the number of
    # columns in the data matrix
    if (length(group) != ncol(data_matrix)) {
        stop("The length of the group variable must match the number of columns in the data matrix.")
    }
    # Ensure the group variable contains only 'normal' and 'tumor'
    if (!all(group %in% c("normal", "tumor"))) {
        stop("The group variable must only contain 'normal' and 'tumor' values.")
    }

    # Calculate the mean for each row in the tumor and normal groups
    mean_tumor <- rowMeans(data_matrix[, group == "tumor"], na.rm = TRUE)
    mean_normal <- rowMeans(data_matrix[, group == "normal"], na.rm = TRUE)

    # Calculate the fold change
    fold_change <- mean_tumor - mean_normal

    # Initialize a vector to store p-values
    p_values <- numeric(nrow(data_matrix))

    # Perform t-test for each row
    for (i in 1:nrow(data_matrix)) {
        normal_values <- data_matrix[i, group == "normal"]
        tumor_values <- data_matrix[i, group == "tumor"]
        wilcox_test_result <- wilcox.test(normal_values, tumor_values,
            paired = FALSE)
        p_values[i] <- wilcox_test_result$p.value
    }

    # Adjust the p-values
    adjusted_p_values <- p.adjust(p_values, method = adjust_method)

    # Create a data frame with fold change, p-values, and adjusted
    # p-values
    results <- data.frame(Fold_Change = fold_change, P_Value = p_values,
        Adjusted_P_Value = adjusted_p_values)
    # Return the results data frame
    return(results)
}

count_zeros_in_rows <- function(mat) {
    # Ensure the input is a matrix
    if (!is.matrix(mat)) {
        stop("Input must be a matrix.")
    }

    # Use rowSums to count zeros in each row
    zero_counts <- rowSums(mat != 0)

    return(zero_counts)
}

Extract and process Methylation data

df <- read.csv("data/omics_regulon_pairs.csv")
labels <- c("kirc", "kirc", "hnsc", "hnsc", "lusc", "lusc", "luad", "luad",
    "paad", "paad")
for (i in c(1, 3, 5, 7, 9)) {
    normal <- read_exp(df$methy[i])
    expr_n <- normal[[1]]
    meta_n <- normal[[2]]

    tumor <- read_exp(df$methy[i+1])
    expr_t <- tumor[[1]]
    meta_t <- tumor[[2]]

    common_terms <- intersect(rownames(expr_t), rownames(expr_n))

    expr_t[!is.finite(as.matrix(expr_t))] <- 0
    expr_n[!is.finite(as.matrix(expr_n))] <- 0

    expr <- cbind(expr_t[common_terms, ], expr_n[common_terms, ])

    saveRDS(expr_n[common_terms, ], paste("output/methy/count_matrix_", labels[i], "_normal.RDS",
        sep = ""))
    saveRDS(expr_t[common_terms, ], paste("output/methy/count_matrix_", labels[i], "_tumor.RDS",
        sep = ""))

    meta <- meta_n[common_terms, ]

    fc <- calculate_log_fold_change_and_pvalue(data_matrix = as.matrix(expr),
        group = c(rep("tumor", ncol(expr_t)), rep("normal", ncol(expr_n))))
    fc <- cbind(meta, fc)
    write.csv(fc, paste("output/DME/", labels[i],
        "_fc.csv", sep = ""), row.names = F)
    fc <- fc[(fc$Adjusted_P_Value < 0.05)&(abs(fc$Fold_Change)>0.1), ]
    write.csv(fc, paste("output/DME/", labels[i],
        "_fc_0.05.csv", sep = ""), row.names = F)
}
binary_unique_index
FALSE  TRUE 
  115 12841 
binary_unique_index
FALSE  TRUE 
  115 12843 
binary_unique_index
FALSE  TRUE 
  115 12842 
binary_unique_index
FALSE  TRUE 
  115 12843 
binary_unique_index
FALSE  TRUE 
  115 12842 
binary_unique_index
FALSE  TRUE 
  115 12842 
binary_unique_index
FALSE  TRUE 
  115 12840 
binary_unique_index
FALSE  TRUE 
  115 12843 
binary_unique_index
FALSE  TRUE 
  115 12842 
binary_unique_index
FALSE  TRUE 
  115 12843 

sessionInfo()
R version 4.4.0 (2024-04-24)
Platform: aarch64-apple-darwin20
Running under: macOS Sonoma 14.5

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/New_York
tzcode source: internal

attached base packages:
[1] stats4    stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
 [1] EnsDb.Hsapiens.v86_2.99.0 ensembldb_2.28.0         
 [3] AnnotationFilter_1.28.0   GenomicFeatures_1.56.0   
 [5] AnnotationDbi_1.66.0      GenomicRanges_1.56.1     
 [7] GenomeInfoDb_1.40.1       IRanges_2.38.0           
 [9] S4Vectors_0.42.0          stringr_1.5.1            
[11] plyr_1.8.9                dplyr_1.1.4              
[13] aracne.networks_1.30.0    viper_1.38.0             
[15] Biobase_2.64.0            BiocGenerics_0.50.0      

loaded via a namespace (and not attached):
  [1] bitops_1.0-7                DBI_1.2.3                  
  [3] rlang_1.1.4                 magrittr_2.0.3             
  [5] git2r_0.33.0                matrixStats_1.3.0          
  [7] e1071_1.7-14                compiler_4.4.0             
  [9] RSQLite_2.3.7               png_0.1-8                  
 [11] vctrs_0.6.5                 ProtGenerics_1.36.0        
 [13] pkgconfig_2.0.3             crayon_1.5.2               
 [15] fastmap_1.2.0               XVector_0.44.0             
 [17] utf8_1.2.4                  Rsamtools_2.20.0           
 [19] promises_1.3.0              rmarkdown_2.27             
 [21] UCSC.utils_1.0.0            purrr_1.0.2                
 [23] bit_4.0.5                   xfun_0.45                  
 [25] zlibbioc_1.50.0             cachem_1.1.0               
 [27] jsonlite_1.8.8              blob_1.2.4                 
 [29] later_1.3.2                 DelayedArray_0.30.1        
 [31] BiocParallel_1.38.0         parallel_4.4.0             
 [33] R6_2.5.1                    bslib_0.7.0                
 [35] stringi_1.8.4               rtracklayer_1.64.0         
 [37] jquerylib_0.1.4             SummarizedExperiment_1.34.0
 [39] Rcpp_1.0.12                 knitr_1.47                 
 [41] mixtools_2.0.0              httpuv_1.6.15              
 [43] Matrix_1.7-0                splines_4.4.0              
 [45] tidyselect_1.2.1            abind_1.4-5                
 [47] rstudioapi_0.16.0           yaml_2.3.8                 
 [49] codetools_0.2-20            curl_5.2.1                 
 [51] lattice_0.22-6              tibble_3.2.1               
 [53] KEGGREST_1.44.0             evaluate_0.24.0            
 [55] survival_3.7-0              proxy_0.4-27               
 [57] kernlab_0.9-32              Biostrings_2.72.1          
 [59] pillar_1.9.0                MatrixGenerics_1.16.0      
 [61] whisker_0.4.1               KernSmooth_2.23-24         
 [63] plotly_4.10.4               generics_0.1.3             
 [65] rprojroot_2.0.4             RCurl_1.98-1.14            
 [67] ggplot2_3.5.1               munsell_0.5.1              
 [69] scales_1.3.0                class_7.3-22               
 [71] glue_1.7.0                  lazyeval_0.2.2             
 [73] tools_4.4.0                 BiocIO_1.14.0              
 [75] data.table_1.15.4           GenomicAlignments_1.40.0   
 [77] fs_1.6.4                    XML_3.99-0.16.1            
 [79] grid_4.4.0                  tidyr_1.3.1                
 [81] colorspace_2.1-0            nlme_3.1-165               
 [83] GenomeInfoDbData_1.2.12     restfulr_0.0.15            
 [85] cli_3.6.2                   workflowr_1.7.1            
 [87] fansi_1.0.6                 S4Arrays_1.4.1             
 [89] segmented_2.1-0             viridisLite_0.4.2          
 [91] gtable_0.3.5                sass_0.4.9                 
 [93] digest_0.6.35               SparseArray_1.4.8          
 [95] rjson_0.2.21                htmlwidgets_1.6.4          
 [97] memoise_2.0.1               htmltools_0.5.8.1          
 [99] lifecycle_1.0.4             httr_1.4.7                 
[101] bit64_4.0.5                 MASS_7.3-61