# HG changeset patch
# User rnateam
# Date 1666253910 0
# Node ID e1cb2e012307912e6d046be5e2ce9d274c838eed
"planemo upload for repository https://github.com/bgruening/galaxytools/tree/master/tools/rna_tools/dewseq commit 71db0e65b3b306904ae2b17ce3de677244aea776"
diff -r 000000000000 -r e1cb2e012307 analyseStudy.Rmd
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/analyseStudy.Rmd	Thu Oct 20 08:18:30 2022 +0000
@@ -0,0 +1,486 @@
+---
+title: "DEWSeq Analysis"
+author: "Thomas Schwarzl and Sudeep Sahadevan"
+date: "07/10/2020"
+output:
+  BiocStyle::html_document:
+    toc: true
+    toc_float: true
+    toc_depth: 5
+params:
+  protein: ""
+  sampleinfo_file: ""
+  countmatrix_file: ""
+  annotation_file: ""
+  output_windows_file: ""
+  output_regions_file: ""
+  output_bed_file: ""
+  output_rdata: ""
+  min_count: 2
+  min_sample: 2
+  LRT: FALSE
+  p_value_cutoff: 0.05
+  lfc_cutoff: 1
+  overlap_correction: FALSE
+  IHW: TRUE
+  decide_fit: TRUE
+---
+```{r setup,  include=FALSE}
+knitr::opts_chunk$set(echo = TRUE)
+```
+
+
+```{r assignData,  echo=FALSE,  eval=TRUE}
+protein <- params$protein
+sampleinfo_file <- params$sampleinfo_file
+countmatrix_file <- params$countmatrix_file
+annotation_file <- params$annotation_file
+output_windows_file <- params$output_windows_file
+output_regions_file <- params$output_regions_file
+output_bed_file <- params$output_bed_file
+output_rdata <- params$output_rdata
+min_count <- params$min_count
+min_sample <- params$min_sample
+p_value_cutoff <- params$p_value_cutoff
+lfc_cutoff <- params$lfc_cutoff
+```
+
+Sanity check input parameter values
+
+```{r sanityCheck,  echo=FALSE,  eval=TRUE}
+# round of min_count and min_sample values first
+message("Any decimals given as values for min_count and min_sample parameters will be rounded off to the nearest integer.")
+min_count <- round(min_count)
+min_sample <- round(min_sample)
+# receive and sanity check p_value_cutoff
+if (p_value_cutoff <= 0 || p_value_cutoff >= 1) {
+  warning("p_value_cutoff must satisfy: 0<=p_value_cutoff<=1. Resetting to default value: 0.05")
+  p_value_cutoff <- 0.05
+}
+# sanity check log2Foldchange cutoff
+if (lfc_cutoff < 0) {
+  warning("lfc_cutoff must be a value >=0. Resetting to default value: 1.00")
+  lfc_cutoff <- 1.0
+}
+# sanity check LRT vs Wald
+if (is(params$LRT,  "logical")) {
+  lrt <- params$LRT
+}else {
+  warning("LRT must be TRUE or FALSE,   setting this parameter to default: FALSE")
+  lrt <- FALSE
+}
+# sanity check overlap correction parameter
+if (is(params$overlap_correction,  "logical")) {
+  overlap_correction <- params$overlap_correction
+}else {
+  warning("overlap_correction must be TRUE or FALSE,  setting this parameter to default: TRUE")
+  overlap_correction <- TRUE
+}
+# sanity check IHW vs BH correction parameter
+if (is(params$IHW, "logical")) {
+  ihw_filt <- params$IHW
+}else {
+  warning("IHW must be TRUE or FALSE,  setting this parameter to default: TRUE")
+  ihw_filt <- TRUE
+}
+# sanity check automated fit vs parametric fit paramter
+if (is(params$decide_fit, "logical")) {
+  decide_fit <- params$decide_fit
+}else {
+  warning("decide_fit must be TRUE or FALSE,  setting this parameter to default: TRUE")
+  decide_fit <- TRUE
+}
+```
+
+# eCLIP analysis of `r protein` 
+
+
+## Setup
+
+This is the analysis of __`r protein`__ with    
+sampleinfo file: ``r sampleinfo_file``   
+countmatrix file: ``r countmatrix_file`` and     
+annotation file: ``r annotation_file`` 
+
+with the following threshold:
+
+minimum read count per window per sample: ``r min_count``
+number of samples with minimum read count per window: ``r min_sample``
+
+using the following parameters:   
+p-value cut-off: ``r p_value_cutoff``  
+Log2FoldChange cut-off: ``r lfc_cutoff``
+use automated method for dispersion estmation: ``r decide_fit``    
+use LRT test : ``r lrt``  
+use overlap correction: ``r overlap_correction``   
+use IHW for FDR correction: ``r ihw_filt``   
+
+
+``` {r check if files exists,  echo=FALSE}
+file_exists <- function(x) {
+  if (!file.exists(x))
+    stop(paste0("'",  x,  "' - file does not exist."))
+}
+
+file_exists(countmatrix_file)
+file_exists(annotation_file)
+file_exists(sampleinfo_file)
+```
+
+First,  we load the libraries.
+
+```{r load DEWSeq}
+required_packages <- c("DEWSeq", "data.table", "IHW", "R.utils", "tidyverse", "ggrepel")
+installed_packages <- installed.packages()[, 1]
+missing_packages <- setdiff(required_packages, installed_packages)
+if (length(missing_packages) != 0) {
+  stop("Found missing dependencies! Please install the following package(s): ", paste(missing_packages, collapse = ", "))
+}
+suppressPackageStartupMessages({
+  require(DEWSeq)
+  require(tidyverse)
+  require(data.table)
+  require(IHW)
+  require(R.utils)
+  require(ggrepel)
+})
+```
+
+## Read in data
+
+Here we read in the window counts
+
+```{r read window counts}
+window_counts <- fread(countmatrix_file,  sep = "\t", stringsAsFactors = FALSE) %>% as.data.frame()
+rownames(window_counts) <- window_counts[, 1]
+window_counts <- window_counts[, -1]
+```
+
+and the sample info file
+
+```{r read sample info}
+sample_info <- read.table(sampleinfo_file, sep = "\t", stringsAsFactors = FALSE)
+if (ncol(sample_info) < 2) {
+  stop("sampleinfo_file ", sampleinfo_file, " MUST have atleast two columns: first column should be the sample names used in ", countmatrix_file,
+       " and second column must be the experiment type: IP or SMI")
+}else if (ncol(sample_info) > 2) {
+  message("Found ", ncol(sample_info), " columns in ", sampleinfo_file, " using the first column as sample name and second column as experiment name")
+  sample_info <- sample_info[, c(1, 2)]
+}
+colnames(sample_info) <- c("samples", "type")
+rownames(sample_info) <- sample_info[, 1]
+```
+
+Now we make sure that the sampleinfo file contains the column "type" with values "SMI" and "IP" only. 
+
+```{r sampleSanity}
+# make sure that sample_info rows and window_counts columns are in same order
+common_samples <- sort(intersect(colnames(window_counts), rownames(sample_info)))
+if (length(common_samples) != ncol(window_counts)) {
+  stop("The number of samples in ", countmatrix_file, " and ", sampleinfo_file, " do not MATCH!")
+}
+sample_info <- sample_info[common_samples,  ]
+window_counts <- window_counts[, common_samples]
+# Now make sure that sample_info$type contains only "IP" and "SMI"
+type_check <- setdiff(unique(sample_info$type), c("IP", "SMI"))
+if (length(type_check) != 0) {
+  stop("The second column in ", sampleinfo_file, " should contain analysis types:'IP' or 'SMI' only. Found unknown value(s): ",
+       paste(type_check, collapse = ",  "))
+}
+```
+
+We make sure that only IP and SMI are in the right factor level order
+
+```{r sampleFactor}
+sample_info <- sample_info %>% mutate(type = factor(type,  levels = c("SMI",  "IP")))
+```
+
+
+
+We create the DEWSeq object
+
+```{r dewseqInit}
+ddw <- DESeqDataSetFromSlidingWindows(countData  = window_counts,
+                                      colData    = sample_info,
+                                      annotObj   = annotation_file,
+                                      tidy       = FALSE,
+                                      design     = ~type)
+```
+
+
+## Prefiltering
+
+```{r prefiltering1}
+# remove all empty windows
+keep <- rowSums(counts(ddw)) >= 1
+ddw <- ddw[keep, ]
+```
+
+
+## Estimating size factors
+
+
+```{r size factors}
+ddw <- estimateSizeFactors(ddw)
+sizeFactors(ddw)
+```
+
+### estimate size factors for only protein_coding genes
+
+```{r protein_coding_size_factors}
+ddw_mrnas <- ddw[rowData(ddw)[, "gene_type"] == "protein_coding",  ]
+ddw_mrnas <- estimateSizeFactors(ddw_mrnas)
+```
+
+### estimate size factors without significant windows
+
+```{r size_factors_no_sig_windows}
+ddw_tmp <- ddw
+ddw_tmp <- estimateDispersions(ddw_tmp,  fitType = "local",  quiet = TRUE)
+if (lrt) {
+  ddw_tmp <- nbinomLRT(ddw_tmp, full = ~type, reduced = ~1)
+}else {
+  ddw_tmp <- nbinomWaldTest(ddw_tmp)
+}
+
+tmp_significant_windows <-
+                results(ddw_tmp,
+                    contrast = c("type",  "IP",  "SMI"),
+                    tidy = TRUE,
+                    filterFun = ihw) %>%
+                dplyr::filter(padj < p_value_cutoff) %>%
+                .[["row"]]
+rm(ddw_tmp)
+```
+
+estimate the size factors without the significant windows.
+
+```{r final_size_factors}
+ddw_mrnas <- ddw_mrnas[!rownames(ddw_mrnas) %in% tmp_significant_windows,  ]
+ddw_mrnas <- estimateSizeFactors(ddw_mrnas)
+```
+
+before thresholding:
+```{r threshold1}
+dim(ddw)
+```
+
+Now threshold the windows read count table.
+```{r threshold2}
+keep_exp <-  which(rowSums(counts(ddw) > min_count) >= min_sample)
+ddw <- ddw[keep_exp, ]
+```
+
+after thresholding:
+```{r threshold3}
+dim(ddw)
+```
+assign size factors
+
+```{r final_assign}
+sizeFactors(ddw) <- sizeFactors(ddw_mrnas)
+rm(list = c("tmp_significant_windows",  "ddw_mrnas"))
+sizeFactors(ddw)
+```
+
+
+## Differential window analysis
+
+### Dispersion estimates
+
+```{r source,  echo = FALSE,  eval = FALSE}
+# source: https://support.bioconductor.org/p/81094/
+```
+We fit parametric and local fit,  and decide the best fit following this [Bioconductor post](https://support.bioconductor.org/p/81094/)
+
+```{r parametric_dispersion}
+parametric_ddw  <- estimateDispersions(ddw,  fitType = "parametric")
+if (decide_fit) {
+  local_ddw  <- estimateDispersions(ddw,  fitType = "local")
+}
+
+```
+
+This is the dispersion estimate for parametric fit
+```{r plot parametric fit,  fig.wide=TRUE}
+plotDispEsts(parametric_ddw,  main = "Parametric fit")
+```
+
+This is the dispersion estimate for local fit,  given automated decision fitting is enabled:
+```{r plot local fit,  fig.wide = TRUE}
+if (decide_fit) {
+  plotDispEsts(local_ddw,  main = "Local fit")
+}
+
+```
+
+This will get the residuals for either fit,  only for automated decision fitting
+```{r residual fit}
+parametric_resid <- na.omit(with(mcols(parametric_ddw), abs(log(dispGeneEst) - log(dispFit))))
+if (decide_fit) {
+  local_resid <- na.omit(with(mcols(local_ddw), abs(log(dispGeneEst) - log(dispFit))))
+  resid_df <- data.frame(residuals = c(parametric_resid, local_resid),
+                         fitType = c(rep("parametric", length(parametric_resid)),
+                                   rep("local", length(local_resid))))
+  summary(resid_df)
+}
+
+```
+
+and we plot histograms of the fits
+
+```{r plot residual histograms,  fig.wide = TRUE}
+if (decide_fit) {
+  ggplot(resid_df, aes(x = residuals, fill = fitType)) +
+    scale_fill_manual(values = c("darkred", "darkblue")) + geom_histogram(alpha = 0.5, position = "identity", bins = 100) +
+    theme_bw()
+}
+
+```
+
+Now,  we will decide for the better fit based on median
+
+```{r choose_fit}
+summary(parametric_resid)
+if (decide_fit) {
+  summary(local_resid)
+  if (median(local_resid) <= median(parametric_resid)) {
+    cat("chosen fitType: local")
+    ddw <- local_ddw
+  }else {
+    cat("chosen fitType: parametric")
+    ddw <- parametric_ddw
+  }
+  rm(local_ddw, parametric_ddw, resid_df, parametric_resid, local_resid)
+}else {
+  ddw <- parametric_ddw
+  rm(parametric_ddw)
+}
+
+```
+
+
+
+### Wald test or LRT
+
+```{r wald_or_LRT}
+if (lrt) {
+  ddw <- nbinomLRT(ddw, full = ~type,  reduced = ~1)
+}else {
+  ddw <- nbinomWaldTest(ddw)
+}
+
+```
+
+
+### Significance testing
+
+```{r sig_windows}
+result_windows <- resultsDEWSeq(ddw,
+                              contrast = c("type",  "IP",  "SMI"),
+                              tidy = TRUE) %>% as_tibble
+
+result_windows
+```
+
+
+### Multiple hypothesis correction with IHW
+
+You might be interested to correct for multiple hypothesis testing with IHW.
+
+Decide on overlap correction based on the parameter `overlap_correction`
+
+```{r ihw}
+if (overlap_correction && ihw_filt) {
+  result_windows[, "p_adj_IHW"] <- adj_pvalues(ihw(pSlidingWindows ~ baseMean,
+                     data = result_windows,
+                     alpha = p_value_cutoff,
+                     nfolds = 10))
+  padj_col <- "p_adj_IHW"
+}else if (!overlap_correction && ihw_filt) {
+  result_windows[, "p_adj_IHW"] <- adj_pvalues(ihw(pvalue ~ baseMean,
+                     data = result_windows,
+                     alpha = p_value_cutoff,
+                     nfolds = 10))
+  padj_col <- "p_adj_IHW"
+}else if (overlap_correction && !ihw_filt) {
+  padj_col <- "pSlidingWindows.adj"
+}else {
+  result_windows[, "p_adj"] <- p.adjust(result_windows$pvalue, method = "BH")
+  padj_col <- "p_adj"
+}
+
+```
+
+Determine significant windows
+
+```{r filter_sig_windows}
+result_windows <- result_windows %>%
+                      mutate(significant = result_windows[, padj_col] < p_value_cutoff)
+sig_windows <- sum(result_windows$significant)
+```
+
+
+`r sig_windows` windows are significant
+
+
+### Combining windows
+
+```{r,  reg1,  message=FALSE,  eval=TRUE, include=FALSE}
+if (sig_windows > 0) {
+ result_regions <- extractRegions(windowRes  = result_windows,  padjCol    = padj_col,  padjThresh = p_value_cutoff,  log2FoldChangeThresh = lfc_cutoff) %>% as_tibble
+}else {
+ message("Cannot find significant windows in this dataset. Try lowering the p-value and log2FoldChange thresholds!")
+}
+```
+
+```{r extractRegion, eval=FALSE}
+if (sig_windows > 0) {
+ result_regions <- extractRegions(windowRes  = result_windows,  padjCol    = padj_col,  padjThresh = p_value_cutoff,  log2FoldChangeThresh = lfc_cutoff) %>% as_tibble
+}
+```
+
+                     
+### Writing Bed file
+
+```{r writing bed file}
+if (sig_windows > 1) {
+ toBED(windowRes = result_windows,  regionRes = result_regions, padjThresh = p_value_cutoff, padjCol   = padj_col,  fileName  = output_bed_file)
+}else {
+ message("This analysis does not have enough <=1 significant windows to create BED file for visualization")
+}
+```
+
+
+## Save Session
+```{r save_data}
+# save enriched windows,  gzip results file if the file suffix is .gz
+if (grepl("\\.gz$", output_windows_file, ignore.case = TRUE)) {
+  gz_out <- gzfile(output_windows_file, "w")
+  write.table(result_windows, file = gz_out, sep = "\t", quote = FALSE, row.names = FALSE, col.names = TRUE)
+  close(gz_out)
+}else {
+  write.table(result_windows, file = output_windows_file, sep = "\t", quote = FALSE, row.names = FALSE, col.names = TRUE)
+}
+# save enriched regions
+if (sig_windows > 0) {
+  if (grepl("\\.gz$", output_regions_file, ignore.case = TRUE)) {
+    gz_out <- gzfile(output_regions_file, "w")
+    write.table(result_regions, file = gz_out, sep = "\t", quote = FALSE, row.names = FALSE, col.names = TRUE)
+    close(gz_out)
+  }else {
+    write.table(result_regions, file = output_regions_file, sep = "\t", quote = FALSE, row.names = FALSE, col.names = TRUE)
+  }
+}
+# save session
+# Warning! session images can be heavy!
+if (nchar(output_rdata) > 5) {
+  save.image(file = output_rdata)
+}
+```
+
+## Session Info
+
+```{r session info}
+sessionInfo()
+```
diff -r 000000000000 -r e1cb2e012307 dewseq.xml
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/dewseq.xml	Thu Oct 20 08:18:30 2022 +0000
@@ -0,0 +1,202 @@
+
+
+    - A sliding window based peak caller for eCLIP/iCLIP data
+    
+        python
+        bioconductor-dewseq
+        r-rmarkdown
+        bioconductor-biocstyle
+        r-tidyverse
+        r-ggrepel
+        bioconductor-ihw
+        r-magick
+    
+
+    
+
+    
+        
+        
+        
+        
+        
+        
+        
+        
+        
+        
+        
+        
+
+        
+
+    
+    
+        
+        
+        
+            (output_options['sig_reg_bed_out'] is True)
+        
+        
+            (output_options['sig_win_reg_bed_out'] is True)
+        
+        
+            (output_options['report_html_out'] is True)
+        
+    
+
+    
+        
+            
+            
+            
+            
+            
+            
+            
+            
+            
+            
+            
+            
+        
+
+        
+            
+            
+            
+            
+            
+            
+            
+        
+
+    
+    
+    
+        
+            @incollection{sahadevan2022pipeline,
+                doi={0.1007/978-1-0716-1851-6_10},
+                url={https://doi.org/10.1007/978-1-0716-1851-6_10},
+                title={A Pipeline for Analyzing eCLIP and iCLIP Data with Htseq-clip and DEWSeq},
+                author={Sahadevan, Sudeep and Sekaran, Thileepan and Schwarzl, Thomas},
+                booktitle={Post-Transcriptional Gene Regulation},
+                pages={189--205},
+                year={2022},
+                publisher={Springer}
+              }
+        
+    
+
diff -r 000000000000 -r e1cb2e012307 dewseq_wrapper.py
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/dewseq_wrapper.py	Thu Oct 20 08:18:30 2022 +0000
@@ -0,0 +1,383 @@
+#!/usr/bin/env python3
+
+import argparse
+import os
+import re
+import shutil
+import subprocess
+
+
+"""
+DEWSeq wrapper script dependencies:
+conda install -c bioconda bioconductor-dewseq
+conda install -c conda-forge r-rmarkdown
+conda install -c bioconda bioconductor-biocstyle
+conda install -c conda-forge r-tidyverse
+conda install -c bioconda r-ggrepel
+conda install -c bioconda bioconductor-ihw
+
+Wrapper for DEWSeq R markdown file:
+https://github.com/EMBL-Hentze-group/DEWSeq_analysis_helpers/tree/master/Parametrized_Rmd
+
+
+Test runs
+=========
+
+# This reports 150 significant regions.
+python dewseq_wrapper.py --annot test-data/windows.exp.txt --matrix test-data/Rbp_count_matrix.exp.txt --info test-data/sample_info.exp.txt --out test_out --ds-pvc 0.5
+
+# Wherease, with LRT, DEWSeq reports just one siginificant region.
+python dewseq_wrapper.py --annot test-data/windows.exp.txt --matrix test-data/Rbp_count_matrix.exp.txt --info test-data/sample_info.exp.txt --out test2_out --ds-pvc 0.5 --ds-use-lrt
+
+"""
+
+################################################################################
+
+
+def setup_argument_parser():
+    """Setup argparse parser."""
+    help_description = """
+    Wrapping DEWSeq R markdown script, to call peak regions on the CLIP-seq data,
+    preprocessed by htseq-clip.
+    """
+    # Define argument parser.
+    p = argparse.ArgumentParser(add_help=False,
+                                prog="dewseq_wrapper.py",
+                                description=help_description,
+                                formatter_class=argparse.MetavarTypeHelpFormatter)
+
+    # Required arguments.
+    p.add_argument("-h", "--help",
+                   action="help",
+                   help="Print help message")
+    p.add_argument("--annot",
+                   dest="in_annot",
+                   type=str,
+                   metavar='str',
+                   required=True,
+                   help="DEWseq annotation file, i.e. windows mapped to IDs table file (output of htseq-clip mapToId)")
+    p.add_argument("--matrix",
+                   dest="in_matrix",
+                   type=str,
+                   metavar='str',
+                   required=True,
+                   help="DEWseq count matrix file (output of htseq-clip createMatrix)")
+    p.add_argument("--info",
+                   dest="in_info",
+                   type=str,
+                   metavar='str',
+                   required=True,
+                   help="DEWseq sample information file (output of htsc_create_count_table.py / htseq-clip Create count table Galaxy wrapper)")
+    p.add_argument("--out",
+                   dest="out_folder",
+                   type=str,
+                   metavar='str',
+                   required=True,
+                   help="Results output folder")
+    p.add_argument("--ds-ms",
+                   dest="ds_ms",
+                   type=int,
+                   metavar='int',
+                   default=2,
+                   help="DEWSeq min_sample parameter. Keep only the windows with at least min_sample number of samples with crosslink site count > min_count (default: 2)")
+    p.add_argument("--ds-mc",
+                   dest="ds_mc",
+                   type=int,
+                   metavar='int',
+                   default=2,
+                   help="DEWSeq min_count parameter. Minimum crosslink site per window per sample (default: 2)")
+    p.add_argument("--ds-pvc",
+                   dest="ds_pvc",
+                   type=float,
+                   metavar='float',
+                   default=0.1,
+                   help="DEWSeq p_value_cutoff parameter. p adjusted value threshold for significant windows (default: 0.1)")
+    p.add_argument("--ds-lfcc",
+                   dest="ds_lfcc",
+                   type=float,
+                   metavar='float',
+                   default=1,
+                   help="DEWSeq lfc_cutoff parameter. Log2 fold change threshold for significant windows (default: 1)")
+    p.add_argument("--ds-oc",
+                   dest="ds_use_oc",
+                   default=False,
+                   action="store_true",
+                   help="DEWSeq overlap_correction parameter. By default FALSE, i.e., do not adjust p-value for overlapping windows. If TRUE use Bonferroni family wise error rate correction on overlapping sliding windows (default: false)")
+    p.add_argument("--ds--disable-ihw",
+                   dest="ds_disable_ihw",
+                   default=False,
+                   action="store_true",
+                   help="Disable DEWSeq IHW parameter. By default, use IHW for multiple tesing correction instead of default BH (Benjamini Hochberg). NOTE: We recommend using IHW instead of default BH for FDR correction")
+    p.add_argument("--ds--disable-df",
+                   dest="ds_disable_df",
+                   default=False,
+                   action="store_true",
+                   help="Disable DEWSeq decide_fit parameter. By default, decide on dispersion estimation fit type local or parametric. If disabled, Use parametric fit. NOTE: decide_fit=TRUE will fit data using both parametric and local fit types and will choose the best fit of the two (see DESeq2 vignette for details). Typically, this should give better results compared to using the default fit type parametric. But, keep in mind that this will also increase the total run time")
+    p.add_argument("--ds-use-lrt",
+                   dest="ds_use_lrt",
+                   default=False,
+                   action="store_true",
+                   help="DEWSeq LRT parameter. Use LRT if the given value is TRUE (see DESeq2 vignette for details). By default, DEWSeq uses Wald test.  NOTE: In our experience, LRT is more accurate than Wald test. But, keep in mind that LRT is a stringent test in comparison to Wald. So if your protein of interest is a very active binder, run the analysis with LRT=TRUE, otherwise use it with caution as you may end up with no significant windows or regions in your final output")
+    p.add_argument("--ds-id",
+                   dest="ds_id",
+                   type=str,
+                   default="RBP",
+                   metavar='str',
+                   help="DEWSeq dataset ID for output report (default: RBP)")
+    p.add_argument("--ds-markdown",
+                   dest="ds_markdown",
+                   type=str,
+                   default="analyseStudy.Rmd",
+                   metavar='str',
+                   help="Provide path to DEWSeq R markdown file. By default assumed to be in working directory")
+    p.add_argument("--copy-md",
+                   dest="copy_markdown",
+                   default=False,
+                   action="store_true",
+                   help="Copy DEWSeq R markdown file to output directory and execute [sic!] it there (default: False)")
+    return p
+
+
+################################################################################
+
+def count_file_rows(in_file,
+                    nr_cols=False):
+    """
+    Count number of file rows. If nr_cols set, demand certain (nr_cols) number
+    of columns (separated by tab), in order for row to be counted.
+
+    """
+    c = 0
+    with open(in_file) as f:
+        for line in f:
+            cols = line.strip().split("\t")
+            if nr_cols:
+                if len(cols) == nr_cols:
+                    c += 1
+            else:
+                c += 1
+    f.closed
+    return c
+
+
+################################################################################
+
+def check_two_dic_same_keys(d1, d2):
+    """
+    Check if two dictionaries have same keys.
+
+    >>> d1 = {'k1': 1, 'k2': 2}
+    >>> d2 = {'k1': 3, 'k2': 4}
+    >>> check_two_dic_same_keys(d1, d2)
+    True
+    >>> d2 = {'k1': 3, 'k3': 4}
+    >>> check_two_dic_same_keys(d1, d2)
+    False
+
+    """
+    assert d1, "given dictionary d1 empty"
+    assert d2, "given dictionary d2 empty"
+    for k in d1:
+        if k not in d2:
+            return False
+    for k in d2:
+        if k not in d1:
+            return False
+    return True
+
+
+################################################################################
+
+if __name__ == '__main__':
+
+    parser = setup_argument_parser()
+    args = parser.parse_args()
+
+    assert os.path.exists(args.in_annot), "--annot file \"%s\" not found" % (args.in_annot)
+    assert os.path.exists(args.in_matrix), "--matrix file \"%s\" not found" % (args.in_matrix)
+    assert os.path.exists(args.in_info), "--info file \"%s\" not found" % (args.in_info)
+    assert os.path.exists(args.ds_markdown), "--ds-markdown file \"%s\" not found" % (args.ds_markdown)
+
+    # Input files.
+    annot_in = os.path.abspath(args.in_annot)
+    matrix_in = os.path.abspath(args.in_matrix)
+    info_in = os.path.abspath(args.in_info)
+    md_in = os.path.abspath(args.ds_markdown)
+
+    # Sum Sanity Checks.
+    matrix_ids = {}
+
+    with open(matrix_in) as f:
+        for line in f:
+            cols = line.strip().split("\t")
+            for c in cols[1:]:
+                matrix_ids[c] = 1
+            break
+    f.closed
+
+    assert matrix_ids, "no dataset columns found in count table file"
+
+    info_ids = {}
+
+    with open(info_in) as f:
+        for line in f:
+            if re.search("^Sample name", line):
+                continue
+            cols = line.strip().split("\t")
+            if cols[0] == "Sample name":
+                continue
+            else:
+                info_ids[cols[0]] = 1
+    f.closed
+
+    assert info_ids, "no dataset columns found in info table file"
+    assert len(matrix_ids) == len(info_ids), "differing numbers of dataset IDs in count table and info table file"
+    assert check_two_dic_same_keys(matrix_ids, info_ids), "dataset IDs in count table and info table file not identical"
+    assert args.ds_ms <= len(matrix_ids), "set DEWSeq min_sample > number of data samples in count / info table files (%i > %i)" % (args.ds_ms, len(matrix_ids))
+    print("Dataset IDs are valid ... ")
+
+    # Output folder.
+    if not os.path.exists(args.out_folder):
+        os.makedirs(args.out_folder)
+
+    # Output files.
+    abs_path_out = os.path.abspath(args.out_folder)
+
+    # Copy markdown file to results folder.
+    if args.copy_markdown:
+        md_source = md_in
+        md_in = abs_path_out + "/analyseStudy.Rmd"
+        if not os.path.exists(md_in):
+            print("Copying markdown file to output folder ... ")
+            shutil.copyfile(md_source, md_in)
+
+    html_out = abs_path_out + "/report.html"
+    win_csv_out = abs_path_out + "/windows.csv"
+    sig_reg_csv_out = abs_path_out + "/significant_regions.csv"
+    sig_win_reg_bed_out = abs_path_out + "/significant_windows_and_regions.bed"
+    sig_reg_bed_out = abs_path_out + "/significant_regions.bed"
+
+    # Delete existing files (as if no peaks found old files would be reported).
+    if os.path.exists(html_out):
+        os.remove(html_out)
+    if os.path.exists(win_csv_out):
+        os.remove(win_csv_out)
+    if os.path.exists(sig_reg_csv_out):
+        os.remove(sig_reg_csv_out)
+    if os.path.exists(sig_win_reg_bed_out):
+        os.remove(sig_win_reg_bed_out)
+    if os.path.exists(sig_reg_bed_out):
+        os.remove(sig_reg_bed_out)
+
+    """
+    Run DEWSeq R markdown file.
+
+    """
+
+    md_ihw = "TRUE"
+    md_df = "TRUE"
+    md_lrt = "FALSE"
+    md_olc = "FALSE"
+    if args.ds_disable_ihw:
+        md_ihw = "FALSE"
+    if args.ds_disable_df:
+        md_df = "FALSE"
+    if args.ds_use_lrt:
+        md_lrt = "TRUE"
+    if args.ds_use_oc:
+        md_olc = "TRUE"
+
+    md_cmd = "Rscript -e 'rmarkdown::render("
+    md_cmd += 'input = "%s", ' % (md_in)
+    md_cmd += 'output_file = "%s", ' % (html_out)
+    md_cmd += 'params = list(protein = "%s", ' % (args.ds_id)
+    md_cmd += 'sampleinfo_file = "%s", ' % (info_in)
+    md_cmd += 'countmatrix_file = "%s", ' % (matrix_in)
+    md_cmd += 'annotation_file = "%s", ' % (annot_in)
+    md_cmd += 'output_windows_file = "%s", ' % (win_csv_out)
+    md_cmd += 'output_regions_file = "%s", ' % (sig_reg_csv_out)
+    md_cmd += 'output_bed_file = "%s", ' % (sig_win_reg_bed_out)
+    md_cmd += 'min_count = %i, ' % (args.ds_mc)
+    md_cmd += 'min_sample = %i, ' % (args.ds_ms)
+    md_cmd += 'p_value_cutoff = %s, ' % (str(args.ds_pvc))
+    md_cmd += 'lfc_cutoff = %s, ' % (str(args.ds_lfcc))
+    md_cmd += 'overlap_correction = %s, ' % (md_olc)
+    md_cmd += 'IHW = %s, ' % (md_ihw)
+    md_cmd += 'decide_fit = %s, ' % (md_df)
+    md_cmd += 'LRT = %s))' % (md_lrt)
+    md_cmd += "'"
+
+    print("Running DEWSeq R markdown file ... ")
+    print(md_cmd)
+    output = subprocess.getoutput(md_cmd)
+    print(output)
+
+    print("")
+    print("Checking for output files ... ")
+    if os.path.exists(html_out):
+        print("FOUND HTML report file \"%s\" ... " % (html_out))
+    else:
+        print("MISSING HTML report file \"%s\" ... " % (html_out))
+    if os.path.exists(win_csv_out):
+        print("FOUND windows CSV file \"%s\" ... " % (win_csv_out))
+    else:
+        print("MISSING windows CSV file \"%s\" ... " % (win_csv_out))
+    if os.path.exists(sig_reg_csv_out):
+        print("FOUND significant regions CSV file \"%s\" ... " % (sig_reg_csv_out))
+    else:
+        print("MISSING significant regions CSV file \"%s\" ... " % (sig_reg_csv_out))
+
+    assert os.path.exists(html_out) and os.path.exists(win_csv_out), "DEWSeq terminated / did no produce any output files. This could be due to too strict filter settings (e.g., min_sample, min_count ... ). Please try again with more relaxed settings"
+    assert os.path.exists(html_out), "output file \"%s\" not found" % (html_out)
+    assert os.path.exists(win_csv_out), "output file \"%s\" not found" % (win_csv_out)
+
+    print("")
+    if not os.path.exists(sig_reg_csv_out):
+        print("WARNING: no significant regions found! (missing \"%s\" file)" % (sig_reg_csv_out))
+    else:
+        assert os.path.exists(sig_win_reg_bed_out), "output file \"%s\" not found" % (sig_win_reg_bed_out)
+        c_sig_reg = count_file_rows(sig_reg_csv_out) - 1
+        print("# significant regions:  %i" % (c_sig_reg))
+        # Save contiguous BED regions in separate file.
+        OUTBED = open(sig_reg_bed_out, "w")
+        with open(sig_reg_csv_out) as f:
+            for line in f:
+                row = line.strip()
+                cols = line.strip().split("\t")
+                if cols[1] == "region_begin":
+                    continue
+                chr_id = cols[0]
+                reg_s = cols[1]
+                reg_e = cols[2]
+                pol = cols[3]
+                win_in_reg = int(cols[4])
+                padj_mean = cols[7]
+                logfc_mean = cols[10]
+                reg_id = cols[12]
+                new_reg_id = reg_id
+                # For regions consisting of > 1 window.
+                if win_in_reg > 1:
+                    new_reg_id = reg_id + "@region"
+                # Print out BED with additional columns (padj, logfc).
+                OUTBED.write("%s\t%s\t%s\t%s\t0\t%s\t%s\t%s\n" % (chr_id, reg_s, reg_e, new_reg_id, pol, padj_mean, logfc_mean))
+
+        f.closed
+        OUTBED.close()
+
+    """
+    Report.
+
+    """
+
+    print("")
+    print("OUTPUT FILES")
+    print("============")
+    print("HTML report:\n%s" % (html_out))
+    print("Windows CSV:\n%s" % (win_csv_out))
+    if os.path.exists(sig_reg_csv_out):
+        print("Significant regions CSV:\n%s" % (sig_reg_csv_out))
+    if os.path.exists(sig_win_reg_bed_out):
+        print("Significant windows + regions BED:\n%s" % (sig_win_reg_bed_out))
+    if os.path.exists(sig_reg_bed_out):
+        print("Significant regions BED:\n%s" % (sig_reg_bed_out))
+    print("")
diff -r 000000000000 -r e1cb2e012307 test-data/Rbp_count_matrix.exp.txt
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/Rbp_count_matrix.exp.txt	Thu Oct 20 08:18:30 2022 +0000
@@ -0,0 +1,3246 @@
+unique_id	Rbp_ctrl_rep1	Rbp_exp_rep1	Rbp_exp_rep2
+TU5001:exon0001W00013	2	2	2
+TU5001:exon0001W00014	2	2	3
+TU5001:exon0001W00015	1	1	1
+TU5001:exon0001W00024	1	1	1
+TU5001:exon0001W00025	1	2	1
+TU5001:exon0001W00026	1	1	2
+TU5001:exon0001W00027	1	1	6
+TU5001:exon0001W00028	1	1	5
+TU5001:exon0001W00031	1	5	6
+TU5001:exon0001W00032	3	4	8
+TU5001:exon0001W00033	5	9	18
+TU5001:exon0001W00034	8	23	29
+TU5001:exon0001W00035	3	18	15
+TU5001:exon0001W00036	3	18	16
+TU5001:exon0001W00037	1	2	3
+TU5001:exon0001W00038	1	1	3
+TU5002:exon0001W00001	6	8	16
+TU5002:exon0001W00002	71	39	116
+TU5002:exon0001W00003	100	65	168
+TU5002:exon0001W00004	97	64	164
+TU5002:exon0001W00005	30	33	63
+TU5003:exon0001W00001	2	6	2
+TU5003:exon0001W00004	1	2	2
+TU5003:exon0001W00005	4	3	2
+TU5003:exon0001W00006	4	3	3
+TU5003:exon0001W00007	4	2	2
+TU5003:exon0001W00008	1	0	1
+TU5003:exon0001W00009	1	0	1
+TU5003:exon0001W00012	1	0	0
+TU5003:exon0001W00013	1	1	1
+TU5003:exon0001W00016	1	2	1
+TU5003:exon0001W00017	1	2	1
+TU5003:exon0001W00018	1	1	1
+TU5003:exon0001W00019	1	1	1
+TU5003:exon0001W00020	1	2	0
+TU5003:exon0001W00024	1	5	1
+TU5003:exon0001W00025	1	2	1
+TU5003:exon0001W00026	1	0	1
+TU5003:exon0001W00030	1	4	3
+TU5003:exon0001W00031	5	8	14
+TU5003:exon0001W00032	7	11	19
+TU5003:exon0001W00033	11	10	16
+TU5003:exon0001W00034	6	3	1
+TU5005:exon0001W00002	1	1	1
+TU5005:exon0001W00003	1	4	5
+TU5005:exon0001W00004	5	11	17
+TU5005:exon0001W00005	5	14	23
+TU5005:exon0001W00006	5	17	20
+TU5005:exon0001W00007	1	15	22
+TU5005:exon0001W00008	2	21	27
+TU5005:exon0001W00009	1	25	25
+TU5005:exon0001W00010	3	18	17
+TU5005:exon0001W00011	2	12	14
+TU5005:exon0001W00012	2	6	10
+TU5005:exon0001W00013	1	2	7
+TU5005:exon0001W00014	1	2	5
+TU5005:exon0001W00046	1	0	0
+TU5005:exon0001W00047	1	1	0
+TU5005:exon0001W00055	4	2	0
+TU5005:exon0001W00056	16	2	6
+TU5005:exon0001W00057	17	2	9
+TU5005:exon0001W00058	11	0	8
+TU5005:exon0001W00059	2	2	4
+TU5005:exon0001W00062	2	1	1
+TU5005:exon0001W00063	2	0	1
+TU5005:exon0001W00081	2	2	2
+TU5005:exon0001W00082	3	3	3
+TU5005:exon0001W00083	1	1	1
+TU5005:exon0001W00084	1	2	1
+TU5005:exon0001W00085	1	3	0
+TU5005:exon0001W00086	2	3	0
+TU5005:exon0001W00087	1	1	0
+TU5005:exon0001W00088	1	1	0
+TU5005:exon0001W00101	1	2	1
+TU5005:exon0001W00102	1	2	1
+TU5005:exon0001W00103	1	0	0
+TU5005:exon0001W00106	2	0	3
+TU5005:exon0001W00107	2	0	3
+TU5005:exon0001W00114	1	7	3
+TU5005:exon0001W00115	2	9	4
+TU5005:exon0001W00116	1	8	2
+TU5005:exon0001W00117	1	2	1
+TU5005:exon0001W00120	1	0	3
+TU5005:exon0001W00121	3	0	2
+TU5005:exon0001W00122	3	0	1
+TU5005:exon0001W00123	3	5	3
+TU5005:exon0001W00124	1	6	4
+TU5005:exon0001W00125	2	6	6
+TU5005:exon0001W00126	3	5	4
+TU5005:exon0001W00127	3	5	3
+TU5005:exon0001W00128	1	12	4
+TU5005:exon0001W00129	1	14	12
+TU5005:exon0001W00130	2	12	14
+TU5005:exon0001W00131	2	11	10
+TU5005:exon0001W00132	2	7	8
+TU5005:exon0001W00133	2	6	5
+TU5005:exon0001W00134	2	7	8
+TU5005:exon0001W00135	1	6	5
+TU5005:exon0001W00136	1	5	4
+TU5005:exon0001W00140	1	0	6
+TU5005:exon0001W00141	1	0	5
+TU5005:exon0001W00142	1	0	2
+TU5005:exon0001W00149	3	0	2
+TU5005:exon0001W00150	5	0	3
+TU5005:exon0001W00151	5	0	2
+TU5005:exon0001W00152	1	0	1
+TU5004:exon0001W00002	1	0	0
+TU5004:exon0001W00003	1	0	0
+TU5004:exon0001W00010	1	0	1
+TU5004:exon0001W00011	1	0	1
+TU5006:exon0001W00001	2	2	1
+TU5006:exon0001W00002	1	2	3
+TU5006:exon0001W00003	1	2	2
+TU5006:exon0001W00004	3	4	4
+TU5006:exon0001W00005	4	4	6
+TU5006:exon0001W00006	33	9	23
+TU5006:exon0001W00007	31	6	18
+TU5006:exon0001W00008	1	1	1
+TU5007:exon0001W00002	1	2	2
+TU5007:exon0001W00003	1	2	2
+TU5007:exon0001W00011	4	4	3
+TU5007:exon0001W00012	4	3	3
+TU5007:exon0001W00013	3	1	3
+TU5007:exon0001W00014	2	5	5
+TU5007:exon0001W00015	3	8	5
+TU5007:exon0001W00016	7	10	11
+TU5007:exon0001W00017	12	13	16
+TU5007:exon0001W00018	13	10	12
+TU5007:exon0001W00019	7	9	6
+TU5007:exon0001W00020	2	4	3
+TU5007:exon0001W00029	1	3	4
+TU5007:exon0001W00030	2	9	5
+TU5007:exon0001W00031	3	8	6
+TU5007:exon0001W00032	1	7	2
+TU5007:exon0001W00033	1	2	0
+TU5007:exon0001W00034	1	2	1
+TU5007:exon0001W00035	3	1	1
+TU5007:exon0001W00036	3	3	1
+TU5008:exon0001W00001	2	1	2
+TU5008:exon0001W00002	3	7	6
+TU5008:exon0001W00003	14	24	49
+TU5008:exon0001W00004	20	27	64
+TU5008:exon0001W00005	20	22	53
+TU5008:exon0001W00006	10	7	18
+TU5008:exon0001W00007	1	1	0
+TU5009:exon0001W00001	3	4	7
+TU5009:exon0001W00002	3	7	9
+TU5009:exon0001W00003	2	17	10
+TU5009:exon0001W00004	2	16	10
+TU5009:exon0001W00005	1	5	4
+TU5009:exon0001W00006	1	4	7
+TU5009:exon0001W00007	1	9	18
+TU5009:exon0001W00008	1	22	34
+TU5009:exon0001W00009	3	26	34
+TU5009:exon0001W00010	18	40	37
+TU5009:exon0001W00011	17	32	31
+TU5009:exon0001W00012	1	1	1
+TU5011:exon0001W00002	2	3	5
+TU5011:exon0001W00003	2	5	6
+TU5010:exon0001W00001	4	4	6
+TU5010:exon0001W00002	4	4	6
+TU5010:exon0001W00008	1	1	1
+TU5010:exon0001W00009	1	1	2
+TU5010:exon0001W00011	1	1	6
+TU5010:exon0001W00012	1	2	5
+TU5010:exon0001W00013	1	1	2
+TU5010:exon0001W00016	1	6	4
+TU5010:exon0001W00017	1	6	4
+TU5010:exon0001W00018	1	5	6
+TU5010:exon0001W00021	3	4	8
+TU5010:exon0001W00022	4	2	3
+TU5010:exon0001W00023	5	3	2
+TU5010:exon0001W00024	2	2	2
+TU5010:exon0001W00025	1	2	1
+TU5010:exon0001W00028	2	0	0
+TU5010:exon0001W00029	3	0	0
+TU5010:exon0001W00030	3	0	0
+TU5010:exon0001W00031	1	1	1
+TU5010:exon0001W00034	1	1	3
+TU5010:exon0001W00035	1	0	0
+TU5010:exon0001W00036	1	0	1
+TU5010:exon0001W00041	10	6	14
+TU5010:exon0001W00042	10	7	20
+TU5010:exon0001W00043	8	7	17
+TU5010:exon0001W00044	3	3	6
+TU5010:exon0001W00045	2	2	1
+TU5012:exon0001W00001	9	31	45
+TU5012:exon0001W00002	9	38	52
+TU5012:exon0001W00003	4	8	13
+TU5012:exon0001W00004	4	6	9
+TU5012:exon0001W00005	2	2	4
+TU5012:exon0001W00011	1	1	0
+TU5012:exon0001W00012	1	1	0
+TU5012:exon0001W00013	1	3	1
+TU5012:exon0001W00016	3	1	0
+TU5012:exon0001W00017	3	1	1
+TU5012:exon0001W00018	3	1	1
+TU5012:exon0001W00019	1	0	0
+TU5012:exon0001W00020	1	0	0
+TU5012:exon0001W00021	2	0	0
+TU5012:exon0001W00022	2	0	0
+TU5012:exon0001W00023	2	0	0
+TU5012:exon0001W00026	1	1	2
+TU5012:exon0001W00027	3	3	3
+TU5012:exon0001W00028	2	2	2
+TU5012:exon0001W00029	1	2	2
+TU5012:exon0001W00032	1	2	3
+TU5012:exon0001W00033	2	3	1
+TU5012:exon0001W00034	3	4	6
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+DEWSeq Analysis
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Sanity check input parameter values
+
## Any decimals given as values for min_count and min_sample parameters will be rounded off to the nearest integer.
+
+
 eCLIP analysis of RBP
+
+
 Setup
+
This is the analysis of RBP with
+sampleinfo file: /home/malong/Repos/dewseq_galaxy/test-data/sample_info.exp.txt
+countmatrix file: /home/malong/Repos/dewseq_galaxy/test-data/Rbp_count_matrix.exp.txt and
+annotation file: /home/malong/Repos/dewseq_galaxy/test-data/windows.exp.txt
+
with the following threshold:
+
minimum read count per window per sample: 2
+number of samples with minimum read count per window: 2
+
using the following parameters:
+p-value cut-off: 0.5
+Log2FoldChange cut-off: 1
+use automated method for dispersion estmation: TRUE
+use LRT test : FALSE
+use overlap correction: FALSE
+use IHW for FDR correction: TRUE
+
First, we load the libraries.
+
requiredPackages <- c('DEWSeq','data.table','IHW','R.utils','tidyverse')
+installedPackages <- installed.packages()[,1]
+diffPackages <- setdiff(requiredPackages,installedPackages)
+if(length(diffPackages)!=0){
+  stop('Found missing dependencies! Please install the following package(s): ',paste(diffPackages,collapse = ", "))
+}
+suppressPackageStartupMessages({
+  require(DEWSeq)
+  require(tidyverse)
+  require(data.table)
+  require(IHW)
+  require(R.utils)
+})
+
+
+
 Read in data
+
Here we read in the window counts
+
WINDOWCOUNTS <- fread(countmatrix_file, sep = "\t",stringsAsFactors = FALSE) %>% as.data.frame()
+rownames(WINDOWCOUNTS) <- WINDOWCOUNTS[,1]
+WINDOWCOUNTS <- WINDOWCOUNTS[,-1]
+
and the sample info file
+
SAMPLEINFO <- read.table(sampleinfo_file,sep="\t",stringsAsFactors = FALSE)
+if(ncol(SAMPLEINFO)<2){
+  stop("sampleinfo_file ",sampleinfo_file," MUST have atleast two columns: first column should be the sample names used in ",countmatrix_file,
+       " and second column must be the experiment type: IP or SMI")
+}else if(ncol(SAMPLEINFO)>2){
+  message("Found ",ncol(SAMPLEINFO)," columns in ",sampleinfo_file," using the first column as sample name and second column as experiment name")
+  SAMPLEINFO <- SAMPLEINFO[,c(1,2)]
+}
+colnames(SAMPLEINFO) <- c("samples","type")
+rownames(SAMPLEINFO) <- SAMPLEINFO[,1]
+
Now we make sure that the sampleinfo file contains the column “type” with values “SMI” and “IP” only.
+
# make sure that SAMPLEINFO rows and WINDOWCOUNTS columns are in same order
+commonSamples <- sort(intersect(colnames(WINDOWCOUNTS),rownames(SAMPLEINFO)))
+if(length(commonSamples)!=ncol(WINDOWCOUNTS)){
+  stop("The number of samples in ",countmatrix_file," and ",sampleinfo_file," do not MATCH!")
+}
+SAMPLEINFO <- SAMPLEINFO[ commonSamples, ]
+WINDOWCOUNTS <- WINDOWCOUNTS[,commonSamples]
+# Now make sure that SAMPLEINFO$type contains only "IP" and "SMI"
+typeCheck <- setdiff(unique(SAMPLEINFO$type),c("IP","SMI"))
+if(length(typeCheck)!=0){
+  stop("The second column in ",sampleinfo_file," should contain analysis types: 'IP' or 'SMI' only. Found unknown value(s): ",
+       paste(typeCheck,collapse=", "))
+}
+
We make sure that only IP and SMI are in the right factor level order
+
SAMPLEINFO <- SAMPLEINFO %>% mutate(type = factor(type, levels = c("SMI", "IP")))
+
We create the DEWSeq object
+
ddw <- DESeqDataSetFromSlidingWindows(countData  = WINDOWCOUNTS,
+                                      colData    = SAMPLEINFO,
+                                      annotObj   = annotation_file,
+                                      tidy       = FALSE,
+                                      design     = ~type)
+
+
+
 Prefiltering
+
# remove all empty windows
+keep <- rowSums(counts(ddw)) >= 1
+ddw <- ddw[keep,]
+
+
+
 Estimating size factors
+
ddw <- estimateSizeFactors(ddw)
+sizeFactors(ddw)
+
## Rbp_ctrl_rep1  Rbp_exp_rep1  Rbp_exp_rep2 
+##     0.6299605     1.2599210     1.3867225
+
+
 estimate size factors for only protein_coding genes
+
ddw_mRNAs <- ddw[ rowData(ddw)[,"gene_type"] == "protein_coding", ]
+ddw_mRNAs <- estimateSizeFactors(ddw_mRNAs)
+
+
+
 estimate size factors without significant windows
+
ddw_tmp <- ddw
+ddw_tmp <- estimateDispersions(ddw_tmp, fitType = "local", quiet = TRUE)
+if(LRT){
+  ddw_tmp <- nbinomLRT(ddw_tmp,full = ~type,reduced = ~1)
+}else{
+  ddw_tmp <- nbinomWaldTest(ddw_tmp)
+}
+
+tmp_significant_windows <- 
+                results(ddw_tmp,
+                    contrast = c("type", "IP", "SMI"),
+                    tidy = TRUE,
+                    filterFun = ihw) %>% 
+                dplyr::filter(padj < p_value_cutoff) %>% 
+                .[["row"]]
+rm(ddw_tmp)
+
estimate the size factors without the significant windows.
+
ddw_mRNAs <- ddw_mRNAs[ !rownames(ddw_mRNAs) %in% tmp_significant_windows, ]
+ddw_mRNAs <- estimateSizeFactors(ddw_mRNAs)
+
before thresholding:
+
dim(ddw)
+
## [1] 3245    3
+
Now threshold the windows read count table.
+
keep_exp <-  which(rowSums(counts(ddw)>minCount)>=minSample)
+ddw <- ddw[keep_exp,]
+
after thresholding:
+
dim(ddw)
+
## [1] 1008    3
+
assign size factors
+
sizeFactors(ddw) <- sizeFactors(ddw_mRNAs)
+rm( list = c("tmp_significant_windows", "ddw_mRNAs"))
+sizeFactors(ddw)
+
## Rbp_ctrl_rep1  Rbp_exp_rep1  Rbp_exp_rep2 
+##     0.6299605     1.2599210     1.3867225
+
+
+
+
 Differential window analysis
+
+
 Dispersion estimates
+
We fit parametric and local fit, and decide the best fit following this Bioconductor post
+
parametric_ddw  <- estimateDispersions(ddw, fitType="parametric")
+
## gene-wise dispersion estimates
+
## mean-dispersion relationship
+
## final dispersion estimates
+
if(decide_fit){
+  local_ddw  <- estimateDispersions(ddw, fitType="local")
+}
+
## gene-wise dispersion estimates
+
## mean-dispersion relationship
+
## final dispersion estimates
+
This is the dispersion estimate for parametric fit
+
plotDispEsts(parametric_ddw, main="Parametric fit")
+

+
This is the dispersion estimate for local fit, given automated decision fitting is enabled:
+
if(decide_fit){
+  plotDispEsts(local_ddw, main="Local fit")
+}
+

+
This will get the residuals for either fit, only for automated decision fitting
+
parametricResid <- na.omit(with(mcols(parametric_ddw),abs(log(dispGeneEst)-log(dispFit))))
+if(decide_fit){
+  localResid <- na.omit(with(mcols(local_ddw),abs(log(dispGeneEst)-log(dispFit))))
+  residDf <- data.frame(residuals=c(parametricResid,localResid),fitType=c(rep("parametric",length(parametricResid)),rep("local",length(localResid))))
+  summary(residDf)
+}
+
##    residuals           fitType         
+##  Min.   : 0.000899   Length:2016       
+##  1st Qu.:17.894458   Class :character  
+##  Median :18.468848   Mode  :character  
+##  Mean   :16.776221                     
+##  3rd Qu.:18.926598                     
+##  Max.   :20.945520
+
and we plot histograms of the fits
+
if(decide_fit){
+  ggplot(residDf, aes(x = residuals, fill = fitType)) + scale_fill_manual(values = c("darkred", "darkblue")) + geom_histogram(alpha = 0.5, position='identity', bins = 100) + theme_bw()
+}
+

+
Now, we will decide for the better fit based on median
+
summary(parametricResid)
+
##     Min.  1st Qu.   Median     Mean  3rd Qu.     Max. 
+##  0.09595 18.18429 18.59938 16.85875 18.92660 19.52154
+
if(decide_fit){
+  summary(localResid)
+  if (median(localResid) <= median(parametricResid)){
+    cat("chosen fitType: local")
+    ddw <- local_ddw
+  }else{
+    cat("chosen fitType: parametric")
+    ddw <- parametric_ddw
+  }
+  rm(local_ddw,parametric_ddw,residDf,parametricResid,localResid)
+}else{
+  ddw <- parametric_ddw
+  rm(parametric_ddw)
+}
+
## chosen fitType: local
+
+
+
 Wald test or LRT
+
if(LRT){
+  ddw <- nbinomLRT(ddw,full = ~type, reduced = ~1)
+}else{
+  ddw <- nbinomWaldTest(ddw)
+}
+
+
+
 Significance testing
+
resultWindows <- resultsDEWSeq(ddw,
+                              contrast = c("type", "IP", "SMI"),
+                              tidy = TRUE) %>% as_tibble
+
+resultWindows
+
## # A tibble: 672 × 20
+##    chromosome   begin   end width strand uniqu…¹ gene_id gene_…² gene_…³ gene_…⁴
+##    <chr>        <dbl> <int> <int> <chr>  <chr>   <chr>   <chr>   <chr>   <chr>  
+##  1 Synechocyst…  2467  2517    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  2 Synechocyst…  2487  2537    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  3 Synechocyst…  2507  2557    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  4 Synechocyst…  2527  2577    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  5 Synechocyst…  2547  2597    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  6 Synechocyst…  2567  2617    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  7 Synechocyst…  2587  2637    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  8 Synechocyst…  2607  2657    50 +      TU5005… TU5005  TU5005  protei… exon   
+##  9 Synechocyst…  2627  2677    50 +      TU5005… TU5005  TU5005  protei… exon   
+## 10 Synechocyst…  2647  2697    50 +      TU5005… TU5005  TU5005  protei… exon   
+## # … with 662 more rows, 10 more variables: Nr_of_region <int>,
+## #   Total_nr_of_region <int>, window_number <int>, baseMean <dbl>,
+## #   log2FoldChange <dbl>, lfcSE <dbl>, stat <dbl>, pvalue <dbl>,
+## #   pSlidingWindows <dbl>, pSlidingWindows.adj <dbl>, and abbreviated variable
+## #   names ¹unique_id, ²gene_name, ³gene_type, ⁴gene_region
+
+
+
 Multiple hypothesis correction with IHW
+
You might be interested to correct for multiple hypothesis testing with IHW.
+
Decide on overlap correction based on the parameter overlap_correction
+
if(overlap_correction & IHW){
+  resultWindows[,"p_adj_IHW"] <- adj_pvalues(ihw(pSlidingWindows ~ baseMean, 
+                     data = resultWindows,
+                     alpha = p_value_cutoff,
+                     nfolds = 10))
+  padjCol <- "p_adj_IHW"
+}else if(!overlap_correction & IHW){
+  resultWindows[,"p_adj_IHW"] <- adj_pvalues(ihw(pvalue ~ baseMean, 
+                     data = resultWindows,
+                     alpha = p_value_cutoff,
+                     nfolds = 10))
+  padjCol <- "p_adj_IHW"
+}else if(overlap_correction & !IHW){
+  padjCol <- "pSlidingWindows.adj"
+}else{
+  resultWindows[,'p_adj'] <- p.adjust(resultWindows$pvalue,method="BH")
+  padjCol <- 'p_adj'
+}
+
## Only 1 bin; IHW reduces to Benjamini Hochberg (uniform weights)
+
Determine significant windows
+
resultWindows <- resultWindows %>% 
+                      mutate(significant = resultWindows[ ,padjCol] < p_value_cutoff)
+sigWindows <- sum(resultWindows$significant)
+
672 windows are significant
+
resultWindows %>%
+   filter(significant) %>% 
+   arrange(desc(log2FoldChange)) %>% 
+   .[["gene_name"]] %>% 
+   unique %>% 
+   head(20)
+
##  [1] "TU5062" "TU5031" "TU5077" "TU5086" "TU5122" "TU5102" "TU5088" "TU5089"
+##  [9] "TU5111" "TU5120" "TU5094" "TU5119" "TU5124" "TU5082" "TU5032" "TU5054"
+## [17] "TU5036" "TU5092" "TU5106" "TU5013"
+
+
+
 Combining windows
+
if(sigWindows>0){
+    resultRegions <- extractRegions(windowRes  = resultWindows, padjCol    = padjCol, padjThresh = p_value_cutoff, log2FoldChangeThresh = lfc_cutoff) %>% as_tibble
+}
+
+
+
 Writing Bed file
+
if(sigWindows>1){
+    toBED(windowRes = resultWindows, regionRes = resultRegions,padjThresh=p_value_cutoff,
+        padjCol   = padjCol, fileName  = output_bed_file)
+}else{
+  message("This analysis does not have enough <=2 significant windows to create BED file for visualization")
+}
+
+
+
+
 Save Session
+
# save enriched windows, gzip results file if the file suffix is .gz
+if(grepl("\\.gz$",output_windows_file,ignore.case = TRUE)){
+  gz_out <- gzfile(output_windows_file,"w")
+  write.table(resultWindows,file=gz_out,sep="\t",quote=FALSE,row.names=FALSE,col.names=TRUE)
+  close(gz_out)
+}else{
+  write.table(resultWindows,file=output_windows_file,sep="\t",quote=FALSE,row.names=FALSE,col.names=TRUE)
+}
+# save enriched regions
+if(sigWindows>0){
+  if(grepl("\\.gz$",output_regions_file,ignore.case = TRUE)){
+    gz_out <- gzfile(output_regions_file,"w")
+    write.table(resultRegions,file=gz_out,sep="\t",quote=FALSE,row.names=FALSE,col.names=TRUE)
+    close(gz_out)
+  }else{
+    write.table(resultRegions,file=output_regions_file,sep="\t",quote=FALSE,row.names=FALSE,col.names=TRUE)
+  }
+}
+# save session
+# Warning! session images can be heavy!
+if(nchar(output_Rdata)>5){
+  save.image( file = output_Rdata)
+}
+
+
+
 Session Info
+
sessionInfo()
+
## R version 4.1.3 (2022-03-10)
+## Platform: x86_64-conda-linux-gnu (64-bit)
+## Running under: Ubuntu 22.04.1 LTS
+## 
+## Matrix products: default
+## BLAS/LAPACK: /home/malong/miniconda3/envs/dewseq/lib/libopenblasp-r0.3.21.so
+## 
+## locale:
+##  [1] LC_CTYPE=de_DE.UTF-8       LC_NUMERIC=C              
+##  [3] LC_TIME=de_DE.UTF-8        LC_COLLATE=de_DE.UTF-8    
+##  [5] LC_MONETARY=de_DE.UTF-8    LC_MESSAGES=de_DE.UTF-8   
+##  [7] LC_PAPER=de_DE.UTF-8       LC_NAME=C                 
+##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
+## [11] LC_MEASUREMENT=de_DE.UTF-8 LC_IDENTIFICATION=C       
+## 
+## attached base packages:
+## [1] stats4    stats     graphics  grDevices utils     datasets  methods  
+## [8] base     
+## 
+## other attached packages:
+##  [1] IHW_1.22.0                  data.table_1.14.2          
+##  [3] forcats_0.5.2               stringr_1.4.1              
+##  [5] dplyr_1.0.10                purrr_0.3.5                
+##  [7] readr_2.1.3                 tidyr_1.2.1                
+##  [9] tibble_3.1.8                ggplot2_3.3.6              
+## [11] tidyverse_1.3.2             DEWSeq_1.8.0               
+## [13] BiocParallel_1.28.3         DESeq2_1.34.0              
+## [15] SummarizedExperiment_1.24.0 Biobase_2.54.0             
+## [17] MatrixGenerics_1.6.0        matrixStats_0.62.0         
+## [19] GenomicRanges_1.46.1        GenomeInfoDb_1.30.0        
+## [21] IRanges_2.28.0              S4Vectors_0.32.4           
+## [23] BiocGenerics_0.40.0         R.utils_2.12.0             
+## [25] R.oo_1.25.0                 R.methodsS3_1.8.2          
+## [27] BiocStyle_2.22.0           
+## 
+## loaded via a namespace (and not attached):
+##  [1] googledrive_2.0.0      colorspace_2.0-3       ellipsis_0.3.2        
+##  [4] XVector_0.34.0         fs_1.5.2               farver_2.1.1          
+##  [7] bit64_4.0.5            AnnotationDbi_1.56.1   fansi_1.0.3           
+## [10] lubridate_1.8.0        xml2_1.3.3             splines_4.1.3         
+## [13] cachem_1.0.6           geneplotter_1.72.0     knitr_1.40            
+## [16] jsonlite_1.8.2         broom_1.0.1            annotate_1.72.0       
+## [19] dbplyr_2.2.1           png_0.1-7              BiocManager_1.30.18   
+## [22] compiler_4.1.3         httr_1.4.4             backports_1.4.1       
+## [25] assertthat_0.2.1       Matrix_1.4-1           fastmap_1.1.0         
+## [28] gargle_1.2.1           cli_3.4.1              htmltools_0.5.3       
+## [31] tools_4.1.3            gtable_0.3.1           glue_1.6.2            
+## [34] GenomeInfoDbData_1.2.7 Rcpp_1.0.9             slam_0.1-50           
+## [37] cellranger_1.1.0       jquerylib_0.1.4        vctrs_0.4.2           
+## [40] Biostrings_2.62.0      xfun_0.33              rvest_1.0.3           
+## [43] lifecycle_1.0.3        XML_3.99-0.11          googlesheets4_1.0.1   
+## [46] zlibbioc_1.40.0        scales_1.2.1           hms_1.1.2             
+## [49] parallel_4.1.3         RColorBrewer_1.1-3     yaml_2.3.5            
+## [52] memoise_2.0.1          sass_0.4.2             stringi_1.7.8         
+## [55] RSQLite_2.2.8          highr_0.9              genefilter_1.76.0     
+## [58] rlang_1.0.6            pkgconfig_2.0.3        bitops_1.0-7          
+## [61] lpsymphony_1.22.0      evaluate_0.17          lattice_0.20-45       
+## [64] labeling_0.4.2         bit_4.0.4              tidyselect_1.1.2      
+## [67] magrittr_2.0.3         bookdown_0.29          R6_2.5.1              
+## [70] generics_0.1.3         DelayedArray_0.20.0    DBI_1.1.3             
+## [73] pillar_1.8.1           haven_2.5.1            withr_2.5.0           
+## [76] survival_3.4-0         KEGGREST_1.34.0        RCurl_1.98-1.9        
+## [79] modelr_0.1.9           crayon_1.5.2           fdrtool_1.2.17        
+## [82] utf8_1.2.2             tzdb_0.3.0             rmarkdown_2.17        
+## [85] locfit_1.5-9.6         grid_4.1.3             readxl_1.4.1          
+## [88] blob_1.2.3             reprex_2.0.2           digest_0.6.29         
+## [91] xtable_1.8-4           munsell_0.5.0          bslib_0.4.0
+
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