Basic RNA-seq analysis for single-end data.

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bulk-rna-seq-pipeline-se
bulk-rna-seq-pipeline-se
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Pipeline to run basic RNA-seq analysis on single-end data.

This is a package of Python and R scripts that enable reading, processing and analysis of Omics' datasets. This package implements the Snakemake management workflow system and is currently implemented to work with the cluster management and job scheduling system SLURM. This snakemake workflow utilizes conda installations to download and use packages for further analysis, so please ensure that you have installed miniconda prior to use.

Questions/issues

Please add an issue to the Omics-QC-pipeline repository. We would appreciate if your issue included sample code/files (as appropriate) so that we can reproduce your bug/issue.

Contributing

We welcome contributors! For your pull requests, please include the following:

  • Sample code/file that reproducibly causes the bug/issue

  • Documented code providing fix

  • Unit tests evaluating added/modified methods.

Use

Locate raw files:

  • After sequencing, your raw fastq files are placed in /path/to/sequencing/files . 
$ cd /path/to/raw/data
$ ls -alh

Check md5sum. 

$ md5sum –c md5sum.txt > md5sum_out.txt

Move your files into the archive to be stored. 

$ mv /path/to/raw/data /path/to/archive

Check md5sum again to ensure your sequencing files are not corrupted. 

$ md5sum –c md5sum.txt > md5sum_out.txt

Clone this Pipeline into your working directory. 

$ git clone https://github.com/ohsu-cedar-comp-hub/Bulk-RNA-seq-pipeline-SE.git

Create a samples/raw directory, and a logs directory in your wdir() . 

$ mkdir logs
$ mkdir samples
$ cd samples
$ mkdir raw

Symbollically link the fastq files of your samples to the wdir/samples/raw directory using a bash script loop in your terminal. 

ls -1 /path/to/data/LIB*gz | while read gz; do
 R=$( basename $gz | cut -d '_' -f 3 | awk '{print $1".fastq.gz"}' )
 echo $R
 ln -s ${gz} ./${R}
done

Upload your metadata file to the data directory, with the correct formatting:

  • Columns should read: StudyID Column2 Column3 ...

  • Each row should be a sample, with subsequent desired information provided (RNA extraction date, etc.)

  • Edit omic_config.yaml to include only columns included in this metadata file:

    • This includes meta_columns_to_plot and pca labels

  • All values in this file should be tab-separated

Edit the omic_config.yaml in your wdir() :

  • Change the project_id to a unique project identifier

  • Add appropriate contrasts based on your samples under the [diffexp][contrasts] section

  • Add the path to your metadata file for the omic_meta_data and samples parameters

  • Change base_dir to your current working directory

  • Ensure you have the correct assembly specified

    • Current options for this are: hg19, hg38.89 (ensembl v89) and hg38.90 (ensembl v90)

Do a dry-run of snakemake to ensure proper execution before submitting it to the cluster (in your wdir). 

$ snakemake -np --verbose

Once your files are symbolically linked, you can submit the job to exacloud via your terminal window. 

$ sbatch submit_snakemake.sh

To see how the job is running, look at your queue. 

$ squeue -u your_username

Directed Acyclic Graph (DAG) of an example workflow including two samples

Example Workflow

Code Snippets

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shell:
    """trimmomatic SE -phred33 {input} {output} ILLUMINACLIP:{params.adapter}:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50"""
SnakeMake Trimmomatic From line 12 of rules/align_rmdp.smk 
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shell:
    """fastqc --outdir  samples/fastqc/{wildcards.sample} --extract  -f fastq {input}"""
SnakeMake FastQC From line 26 of rules/align_rmdp.smk 
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shell:
    """fastq_screen --aligner bowtie2 --conf {params.conf} --outdir samples/fastqscreen/{wildcards.sample} {input}"""
SnakeMake Bowtie 2 From line 41 of rules/align_rmdp.smk 
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run:
     STAR=config["star_tool"],
     pathToGenomeIndex = config["star_index"]

     shell("""
            {STAR} --runThreadN {threads} --runMode alignReads --genomeDir {pathToGenomeIndex} \
            --readFilesIn {input} \
            --outFileNamePrefix samples/star/{wildcards.sample}_bam/ \
            --sjdbGTFfile {params.gtf} --quantMode GeneCounts \
            --sjdbGTFtagExonParentGene gene_name \
            --outSAMtype BAM SortedByCoordinate \
            #--readFilesCommand zcat \
            --twopassMode Basic
            """)
SnakeMake From line 56 of rules/align_rmdp.smk 
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shell:
    """samtools index {input} {output}"""
SnakeMake SAMtools From line 79 of rules/align_rmdp.smk 
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script:
    "../scripts/compile_star_log.py"
SnakeMake From line 88 of rules/align_rmdp.smk 
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script:
    "../scripts/compile_star_counts.py"
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script:
    "../scripts/RNAseq_filterCounts.R"
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script:
    "../scripts/deseq2-init.R"
SnakeMake DESeq2 From line 18 of rules/deseq.smk 
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script:
    "../scripts/deseq2_pairwise.R"
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script:
    "../scripts/deseq2_group.R"
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script:
    "../scripts/QC.R"
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script:
    "../scripts/qplot.R"
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script:
    "../scripts/density_plot.R"
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script:
    "../scripts/runGOforDESeq2.R"
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script:
    "../scripts/RNAseq_makeVolcano.R"
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script:
    "../scripts/permutation_test.R"
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script:
    "../scripts/run_glimma.R"
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script:
    "../scripts/run_glimma_mds.R"
SnakeMake From line 198 of rules/deseq.smk 
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shell:
    "insertion_profile.py -s SE -i {input} -o rseqc/insertion_profile/{wildcards.sample}/{wildcards.sample}"
SnakeMake From line 11 of rules/omic_qc.smk 
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shell:
    "clipping_profile.py -i {input} -s SE -o rseqc/clipping_profile/{wildcards.sample}/{wildcards.sample}"
SnakeMake From line 24 of rules/omic_qc.smk 
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shell:
    "read_distribution.py -i {input} -r {params.bed} > {output}"
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shell:
    "read_GC.py -i {input} -o rseqc/read_GC/{wildcards.sample}/{wildcards.sample}"
SnakeMake From line 50 of rules/omic_qc.smk 
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import pandas as pd


"""Function accepts a STAR output directory and compiles all sample information from ReadsPerGene.out.tab

Args:
    snakemake.input (list): list of globbed wildcards STAR ReadsPerGene.out.tab
    project_title (str): Project title for compiled STAR counts
Returns:
    Compiled STAR counts as tab delimited file.
"""
colnames = snakemake.params.samples
tables = [pd.read_csv(fh, header=None, skiprows=4, usecols=[0,3], index_col=0, sep = '\t',  names = ['Genes',fh.split('/')[-2].split('_')[0]]) for fh in snakemake.input]
joined_table = pd.concat(tables, axis=1)
joined_table.columns = colnames
joined_table_sorted = joined_table.reindex(sorted(joined_table.columns), axis = 1)
joined_table_sorted.to_csv(snakemake.output[0], sep='\t')
Python Pandas STAR From line 1 of scripts/compile_star_counts.py 
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import pandas as pd


"""Function accepts a STAR output directory and compiles all sample information from Log.final.out
Args:
    snakemake.input (list): list of globbed wildcards STAR Log.final.out
    project_title (str): Project title for compiled STAR mapping statistics

Returns:
    Compiled STAR log.final.out as tab delimited file.
"""

tables = [pd.read_csv(fh, sep = '\t', index_col = 0, names = [fh.split('/')[-2]]) for fh in snakemake.input]
joined_table = pd.concat(tables, axis=1)
joined_table_sorted = joined_table.reindex(sorted(joined_table.columns), axis = 1)
joined_table_sorted.to_csv(snakemake.output[0], sep='\t')
Python Pandas STAR From line 1 of scripts/compile_star_log.py 
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library(DESeq2)
library(ggplot2)
library(reshape2)
library(data.table)
library(plyr)
library(RColorBrewer)


rld <- snakemake@input[['rld']]
cat(sprintf(c('rld: ', rld, '\n')))


condition <- snakemake@params[['linear_model']]
cat(sprintf(c('condition: ', condition, '\n')))

project_id <- snakemake@params[['project_id']]

density_plot <- snakemake@output[['density']]
cat(sprintf(c('Density plot : ', density_plot, '\n')))


colors <- snakemake@params['colors']
discrete <- snakemake@params['discrete']

# function to grab the ggplot2 colours
gg_color_hue <- function(n) {
  hues = seq(15, 375, length = n + 1)
  hcl(h = hues, l = 65, c = 100)[1:n]
}

rld = readRDS(rld)
normed_values = assay(rld)
normed_t = t(normed_values)
meta = colData(rld)

if(colors[[1]] !='NA' & discrete[[1]] =='NA'){
    if(brewer.pal.info[colors[[1]],]$maxcolors >= length(levels(meta[[condition]]))) {
        pal <- brewer.pal(length(levels(meta[[condition]])),name=colors[[1]])
    } 
} else if(discrete[[1]] != 'NA' & length(discrete)==length(levels(meta[[condition]]))){
        pal <- unlist(discrete)
} else {
        pal <- gg_color_hue(length(levels(meta[[condition]])))
}

joined_counts = cbind(meta[condition],normed_t)

x = as.data.table(joined_counts)
mm <- melt(x,id=condition)

mu <- ddply(mm, condition, summarise, grp.mean=mean(value))
pdf(density_plot)
p<-ggplot(mm, aes_string(x='value', color=condition)) +
  geom_density()+
  geom_vline(data=mu, aes_string(xintercept='grp.mean', color=condition),
             linetype="dashed") + xlab('regularized log expression') + 
  scale_color_manual(values=pal) +
  ggtitle(eval(project_id)) + theme(plot.title = element_text(hjust = 0.5))
p
dev.off()
R ggplot2 data.table RColorBrewer reshape2 plyr From line 1 of scripts/density_plot.R 
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library("DESeq2")
library("ggplot2")
library("pheatmap")
library("dplyr")
library("vsn")
library("RColorBrewer")
library("genefilter")

cat(sprintf(c('Working directory',getwd())))

cat(sprintf('Setting parameters'))

pca_plot <- snakemake@output[['pca']]
cat(sprintf(c('PCA plot: ',pca_plot)))

labels <- snakemake@params[['pca_labels']]
cat(sprintf(c('PCA Labels: ',labels)))

sd_mean_plot <- snakemake@output[['sd_mean_plot']]
cat(sprintf(c('SD Mean plot: ',sd_mean_plot,'\n')))

distance_plot <- snakemake@output[['distance_plot']]
cat(sprintf(c('Distance plot: ',distance_plot,'\n')))

heatmap_plot <- snakemake@output[['heatmap_plot']]
cat(sprintf(c('Heatmap Plot: ', heatmap_plot, '\n')))

rds_out <- snakemake@output[['rds']]
cat(sprintf(c('RDS Output: ', rds_out, '\n')))

rld_out <- snakemake@output[['rld_out']]
cat(sprintf(c('RLD Output: ', rld_out, '\n')))

counts <- snakemake@input[['counts']]
cat(sprintf(c('Counts table: ', counts, '\n')))

metadata <- snakemake@params[['samples']]
cat(sprintf(c('Metadata: ', metadata, '\n')))

sampleID <- snakemake@params[['sample_id']]
cat(sprintf(c('Sample ID: ', sampleID, '\n')))

Type <- snakemake@params[['linear_model']]
cat(sprintf(c('Linear Model: ', Type, '\n')))

group <- snakemake@params[['LRT']]
cat(sprintf(c('Subsetted group: ', group, '\n')))

plot_cols <- snakemake@config[['meta_columns_to_plot']]
subset_cols = names(plot_cols)

# color palette
colors <- snakemake@params[['colors']]
discrete <- snakemake@params[['discrete']]

# function to grab the ggplot2 colours
gg_color_hue <- function(n) {
  hues = seq(15, 375, length = n + 1)
  hcl(h = hues, l = 65, c = 100)[1:n]
}

Dir <- "results/diffexp/group/"

md <- read.delim(file=metadata, sep = "\t", stringsAsFactors = FALSE)
md <- md[order(md[sampleID]),]

# Read in counts table
cts <- read.table(counts, header=TRUE, row.names=1, sep="\t", check.names=F)
cts <- cts[,order(colnames(cts))]

# Put sample IDs as rownames of metadata
rownames(md) <- md[[sampleID]]
md[[sampleID]] <- NULL

# Ensure that we subset md to have exactly the same samples as in the counts table
md <- md[colnames(cts),]
dim(md)

# Check
stopifnot(rownames(md)==colnames(cts))

# Define colours based on number of Conditions
if(colors[[1]] !='NA' & discrete[[1]] =='NA'){
    if (brewer.pal.info[colors[[1]],]$maxcolors >= length(unique(md[[Type]]))) {
        pal <- brewer.pal(length(unique(md[[Type]])),name=colors[[1]])
    } 
} else if(discrete[[1]] != 'NA' & length(discrete)==length(unique(md[[Type]]))){
        pal <- unlist(discrete)
} else {
        pal <- gg_color_hue(length(unique(md[[Type]])))
}

# Create dds object from counts data and correct columns
dds <- DESeqDataSetFromMatrix(countData=cts,
                              colData=md,
                              design= as.formula(paste('~',Type)))

# Remove uninformative columns
dds <- dds[ rowSums(counts(dds)) >= 1, ]

# Likelihood Ratio test to look at differential expression across ALL types, and not just pairs of types (contrast)
dds.lrt <- DESeq(dds, test="LRT", reduced=~1)
res.lrt <- results(dds.lrt, cooksCutoff = Inf, independentFiltering=FALSE)
head(res.lrt)

# Obtain normalized counts
rld <- rlog(dds.lrt, blind=FALSE)

# Pairwise PCA Plot
pdf(pca_plot)
plotPCA(rld, intgroup=labels[[1]])
dev.off()

# Pairwise PCA Plot with more than one PCA parameter
if (length(labels)>1) {
  pca_plot2 <- sub("$","twoDimensional_pca_plot.pdf", Dir)
  pcaData <- plotPCA(rld, intgroup=c(labels[[1]], labels[[2]]), returnData=TRUE)
  pdf(pca_plot2, 5, 5)
  percentVar <- round(100 * attr(pcaData, "percentVar"))
  ggplot(pcaData, aes_string("PC1", "PC2", color=labels[[1]], shape=labels[[2]])) +
    geom_point(size=3) +
    xlab(paste0("PC1: ",percentVar[1],"% variance")) +
    ylab(paste0("PC2: ",percentVar[2],"% variance")) +
    coord_fixed()
  dev.off()
}

# SD mean plot
pdf(sd_mean_plot)
meanSdPlot(assay(rld))
dev.off()

# Heatmap of distances
pdf(distance_plot)
sampleDists <- dist(t(assay(rld)))
sampleDistMatrix <- as.matrix(sampleDists)

rownames(sampleDistMatrix) <- colnames(rld)
colors <- colorRampPalette( rev(brewer.pal(9, "Blues")) )(255)
pheatmap(sampleDistMatrix, fontsize=5, scale="row",
         clustering_distance_rows=sampleDists,
         clustering_distance_cols=sampleDists,
         col=colors)
dev.off()

# Heatmap across all samples
# List top 50 genes for group comparisons
topGenes <- head(order(res.lrt$padj), 50)

# Extract topGenes from rld object
plot <- assay(rld)[topGenes,] #for 2+ types

# Generate data frame with samples as the rownames and single colData as the first row
# Default when we subset creates an incompatible dataframe so this is a check
df <- as.data.frame(colData(rld))
if (length(subset_cols)==1) {
  annot <- as.data.frame(cbind(rownames(df), paste(df[[subset_cols[1]]])))
  names(annot) <- c("SampleID", subset_cols[1])
  rownames(annot) <- annot[[sampleID]]
  annot[[sampleID]] <- NULL
} else {
  annot <- df[,subset_cols]
}

pdf(heatmap_plot)
pheatmap(assay(rld)[topGenes,], cluster_rows=T, scale="row", fontsize=6,fontsize_row=6,fontsize_col=6,show_rownames=T, cluster_cols=T, annotation_col=annot, labels_col=as.character(rownames(df)), main = paste("Heatmap of top 50 DE genes across all samples"))
dev.off()

saveRDS(dds, file=rds_out)
saveRDS(rld, file=rld_out)

group <- as.vector(group)

# If LRT group has been specified, run the analysis for that group
if (length(group)>0) {
  md <- read.delim(file=metadata, sep = "\t", stringsAsFactors = FALSE)
  md <- md[order(md[sampleID]),]
  cts <- read.table(counts, header=TRUE, row.names=1, sep="\t")
  cts <- cts[,order(colnames(cts))]
  md <- md[md[[Type]] %in% group,]
  rownames(md) <- md[[sampleID]]
  md[[sampleID]] <- NULL
  keep <- colnames(cts)[colnames(cts) %in% rownames(md)]
  cts <- cts[, keep]
  dim(cts)
  md <- md[colnames(cts),]
  dim(md)

  dds <- DESeqDataSetFromMatrix(countData=cts,
                              colData=md,
                              design= as.formula(paste('~',Type)))
  dds <- dds[ rowSums(counts(dds)) >= 1, ]
  dds.lrt <- DESeq(dds, test="LRT", reduced=~1)
  res.lrt <- results(dds.lrt, cooksCutoff = Inf, independentFiltering=FALSE)
  rld <- rlog(dds.lrt, blind=FALSE)

  # Pairwise PCA Plot
  pdf(sub("$", "subsetted_pca_plot.pdf", Dir), 5, 5)
  plotPCA(rld, intgroup=labels[[1]])
  dev.off()
  # Pairwise PCA Plot with more than one PCA parameter
  if (length(labels)>1) {
    pcaData <- plotPCA(rld, intgroup=c(labels[[1]], labels[[2]]), returnData=TRUE)
    pdf(sub("$", "subsetted_twoDimensional_pca_plot.pdf", Dir), 5, 5)
    percentVar <- round(100 * attr(pcaData, "percentVar"))
    ggplot(pcaData, aes_string("PC1", "PC2", color=labels[[1]], shape=labels[[2]])) +
      geom_point(size=3) +
      xlab(paste0("PC1: ",percentVar[1],"% variance")) +
      ylab(paste0("PC2: ",percentVar[2],"% variance")) +
      coord_fixed()
    dev.off()
  }

  # Heatmap
  topGenes <- head(order(res.lrt$padj), 50)
  # Extract topGenes from rld object
  plot <- assay(rld)[topGenes,] #for 2+ types

  df <- as.data.frame(colData(rld))
  if (length(subset_cols)==1) {
    annot <- as.data.frame(cbind(rownames(df), paste(df[[subset_cols[1]]])))
    names(annot) <- c("SampleID", subset_cols[1])
    rownames(annot) <- annot[[sampleID]]
    annot[[sampleID]] <- NULL
  } else {
    annot <- df[,subset_cols]
  }

  pdf(sub("$", "subsetted_heatmap.pdf", Dir), 5, 5)
  pheatmap(assay(rld)[topGenes,], cluster_rows=T, scale="row", fontsize=6,fontsize_row=6,fontsize_col=6,show_rownames=T, cluster_cols=T, annotation_col=annot, labels_col=as.character(rownames(df)), main = paste("Heatmap of top 50 DE genes across selected samples"))
  dev.off()
}
R ggplot2 dplyr DESeq2 RColorBrewer PCAtools pheatmap genefilter vsn From line 1 of scripts/deseq2_group.R 
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library("dplyr")
library("DESeq2")

counts = snakemake@input[['counts']]

metadata <- snakemake@params[['samples']]

sampleID <- snakemake@params[['sample_id']]

Type <- snakemake@params[['linear_model']]

contrast <- snakemake@params[['contrast']]

baseline <- contrast[[2]]

target <- contrast[[1]]

output = snakemake@output[['rds']]

rld_out = snakemake@output[['rld_out']]

parallel <- FALSE
if (snakemake@threads > 1) {
    library("BiocParallel")
    # setup parallelization
    register(MulticoreParam(snakemake@threads))
    parallel <- TRUE
}

# Read in metadata table and order according to sampleID
md <- read.delim(file=metadata, sep = "\t", stringsAsFactors = FALSE)
md <- md[order(md[sampleID]),]

# Read in counts table
subdata <- read.table(counts, header=TRUE, row.names=1, sep="\t", check.names=FALSE)
subdata <- subdata[,order(colnames(subdata))]

# Extract only the Types that we want in further analysis & only the PP_ID and Status informative columns
md <- filter(md, !!as.name(Type) == baseline | !!as.name(Type) == target, !!as.name(sampleID) %in% colnames(subdata))

# Keep only the PP_IDs of the types we have chosen in the metadata table above
rownames(md) <- md[[sampleID]]
md[[sampleID]] <- NULL
keep <- colnames(subdata)[colnames(subdata) %in% rownames(md)]
subdata <- subdata[, keep]
dim(subdata)

# Check
stopifnot(rownames(md)==colnames(subdata))

# Obtain the number of genes that meet padj<0.01 for reference line in histogram
dds <- DESeqDataSetFromMatrix(countData=subdata,
                              colData=md,
                              design= as.formula(paste('~',Type)))

dds <- estimateSizeFactors(dds)

# Remove uninformative columns
dds <- dds[ rowSums(counts(dds)) >= 1, ]

# Normalization and pre-processing
dds <- DESeq(dds, parallel=parallel)

saveRDS(dds, file=output)

# colData and countData must have the same sample order, but this is ensured
# by the way we create the count matrix
dds <- dds[ rowSums(counts(dds)) > 1, ]
# normalization and preprocessing
dds <- DESeq(dds, parallel=parallel)
saveRDS(dds, file=output)

# obtain normalized counts
rld <- rlog(dds, blind=FALSE)
saveRDS(rld, file=rld_out)
R dplyr DESeq2 BiocParallel From line 1 of scripts/deseq2-init.R 
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library("DESeq2")
library("pheatmap")
library("ggplot2")
library("ggrepel")

print('Setting parameters')

rds = snakemake@input[['rds']]
cat(sprintf(c('RDS object: ',rds,'\n')))

rld = snakemake@input[['rld']]
cat(sprintf(c('RLD object: ',rld,'\n')))

Type = snakemake@params[['linear_model']]
cat(sprintf(c('Type: ',Type,'\n')))

sampleID = snakemake@params[['sample_id']]
cat(sprintf(c('Sample ID: ',sampleID,'\n')))

ma_plot = snakemake@output[['ma_plot']]
cat(sprintf(c('MA plot', ma_plot,'\n')))

out_table = snakemake@output[['table']]
cat(sprintf(c('Summary results table', out_table,'\n')))

panel_ma = snakemake@output[['panel_ma']]
cat(sprintf(c('MA panel', panel_ma,'\n')))

heatmap_plot = snakemake@output[['heatmap_plot']]
cat(sprintf(c('Heatmap plot', heatmap_plot,'\n')))

var_heat = snakemake@output[['var_heat']]
cat(sprintf(c('Variance Heatmap plot', var_heat,'\n')))

pca_plot = snakemake@output[['pca_plot']]
cat(sprintf(c('PCA plot', pca_plot,'\n')))

labels <- snakemake@params[['pca_labels']]
cat(sprintf(c('PCA Labels: ',labels)))

cat(sprintf('Load dds DESeqTransform object'))
dds <- readRDS(rds)

cat(sprintf('Load rlog DESeqTransform object'))
rld <- readRDS(rld)

Dir <- "results/diffexp/pairwise/"

plot_cols <- snakemake@config[['meta_columns_to_plot']]
subset_cols = names(plot_cols)

contrast <- c(Type, snakemake@params[["contrast"]])
baseline <- contrast[[3]]
target <- contrast[[2]]

upCol = "#FF9999"
downCol = "#99CCFF"
ncCol = "#CCCCCC"
adjp <- 0.01
FC <- 2

parallel <- FALSE
if (snakemake@threads > 1) {
    library("BiocParallel")
    # setup parallelization
    register(MulticoreParam(snakemake@threads))
    parallel <- TRUE
}

# Pairwise PCA Plot
pdf(pca_plot)
plotPCA(rld, intgroup=labels[[1]])
dev.off()

# Pairwise PCA Plot with more than one PCA parameter
if (length(labels)>1) {
  pca_plot2 <- sub("$",paste(contrast[2],"vs",contrast[3],"twoDimensional_pca_plot.pdf", sep = "-"), Dir)
  pcaData <- plotPCA(rld, intgroup=c(labels[[1]], labels[[2]]), returnData=TRUE)
  pdf(pca_plot2, 5, 5)
  percentVar <- round(100 * attr(pcaData, "percentVar"))
  ggplot(pcaData, aes_string("PC1", "PC2", color=labels[[1]], shape=labels[[2]])) +
    geom_point(size=3) +
    xlab(paste0("PC1: ",percentVar[1],"% variance")) +
    ylab(paste0("PC2: ",percentVar[2],"% variance")) +
    coord_fixed()
  dev.off()
}

res <- results(dds, contrast=contrast,  independentFiltering = FALSE, cooksCutoff = Inf)
# shrink fold changes for lowly expressed genes
res <- lfcShrink(dds, contrast=contrast, res=res)

# MA plot - calc norm values yourself to plot with ggplot
# MA plot is log2normalized counts (averaged across all samples) vs. log2FC

# extract normalized counts to calculate values for MA plot
norm_counts <- counts(dds, normalized=TRUE)

## select up regulated genes
forPlot <- as.data.frame(res)
forPlot$log2Norm <- log2(rowMeans(norm_counts))
forPlot$Gene <- rownames(forPlot)

up <- forPlot$padj < adjp & forPlot$log2FoldChange > log2(FC)
sum(up)

## select down regulated genes
down <- forPlot$padj < adjp & forPlot$log2FoldChange < -log2(FC)
sum(down)

# Grab the top 5 up and down regulated genes to label in the volcano plot
if (sum(up)>5) {
  temp <- forPlot[up,]
  upGenesToLabel <- head(rownames(temp[order(-temp$log2FoldChange),], 5))
} else if (sum(up) %in% 1:5) {
  temp <- forPlot[up,]
  upGenesToLabel <- rownames(temp[order(-temp$log2FoldChange),])
}

if (sum(down)>5) {
  temp <- forPlot[down,]
  downGenesToLabel <- head(rownames(temp[order(temp$log2FoldChange),], 5))
} else if (sum(down) %in% 1:5) {
  temp <- forPlot[down,]
  downGenesToLabel <- rownames(temp[order(temp$log2FoldChange),])
}

forPlot$Expression <- ifelse(down, 'down',
                  ifelse(up, 'up','NS'))
forPlot$Expression <- factor(forPlot$Expression, levels=c("up","down","NS"))

# Assign colours to conditions
if (sum(up)==0 & sum(down)==0) {
  colours <- ncCol
} else if (sum(up)==0) {
  colours <- c(downCol, ncCol)
} else if (sum(down)==0) {
  colours <- c(upCol, ncCol)
} else {
  colours <- c(upCol, downCol, ncCol)
}

# Create vector for labelling the genes based on whether genes are DE or not
if (exists("downGenesToLabel") & exists("upGenesToLabel")) {
  genesToLabel <- c(downGenesToLabel, upGenesToLabel)
} else if (exists("downGenesToLabel") & !exists("upGenesToLabel")) {
  genesToLabel <- downGenesToLabel
} else if (!exists("downGenesToLabel") & exists("upGenesToLabel")) {
  genesToLabel <- upGenesToLabel
}

if (exists("genesToLabel")) {
  maPlot <- ggplot(forPlot, mapping=aes(x=log2Norm, y=log2FoldChange, colour=Expression)) +
    geom_point() +
    geom_hline(yintercept=c(-1,1), linetype="dashed", color="black") +
    geom_label_repel(aes(label=ifelse(Gene %in% genesToLabel, as.character(Gene),'')),box.padding=0.1, point.padding=0.5, segment.color="gray70", show.legend=FALSE) +
    scale_colour_manual(values=colours) +
    ggtitle(paste(baseline, "vs", target)) +
    xlab("log2(Normalized counts)") +
    ylab("log2(Fold Change)") +
    theme(plot.title = element_text(hjust=0.5))
} else {
  maPlot <- ggplot(forPlot, mapping=aes(x=log2Norm, y=log2FoldChange, colour=Expression)) +
    geom_point() +
    geom_hline(yintercept=c(-1,1), linetype="dashed", color="black") +
    scale_colour_manual(values=colours) +
    ggtitle(paste(baseline, "vs", target)) +
    xlab("log2(Normalized counts)") +
    ylab("log2(Fold Change)") +
    theme(plot.title = element_text(hjust=0.5))
}

# MA plot
pdf(ma_plot)
print({
  maPlot
})
dev.off()

# P-histogram
p_hist = snakemake@output[['p_hist']]
pdf(p_hist)
hist(res$pvalue[res$baseMean > 1], breaks = 0:20/20, col = "grey50", border = "white", main='P values for genes with mean normalized count larger than 1',xlab='pvalue')
dev.off()

#panel ma plot
pdf(panel_ma)
par(mfrow=c(2,2),mar=c(2,2,1,1) +0.1)
ylim <- c(-2.5,2.5)
resGA <- results(dds, contrast=contrast, lfcThreshold=.5, altHypothesis="greaterAbs")
resLA <- results(dds, contrast=contrast, lfcThreshold=.5, altHypothesis="lessAbs")
resG <- results(dds, contrast=contrast, lfcThreshold=.5, altHypothesis="greater")
resL <- results(dds, contrast=contrast, lfcThreshold=.5, altHypothesis="less")
drawLines <- function() abline(h=c(-.5,.5),col="dodgerblue",lwd=2)
plotMA(resGA, ylim=ylim); drawLines()
plotMA(resLA, ylim=ylim); drawLines()
plotMA(resG, ylim=ylim); drawLines()
plotMA(resL, ylim=ylim); drawLines()
mtext(resG@elementMetadata$description[[2]], outer=T, cex=.6,line=-1)
dev.off()

# Heatmap of top 50 genes
topGenes <- head(order(res$padj),50)

df <- as.data.frame(colData(rld))
if (length(subset_cols)==1) {
  annot <- as.data.frame(cbind(rownames(df), paste(df[[subset_cols[1]]])))
  names(annot) <- c("SampleID", subset_cols[1])
  rownames(annot) <- annot$SampleID
  annot$SampleID <- NULL
} else {
  annot <- df[,subset_cols]
}

pdf(heatmap_plot)
pheatmap(assay(rld)[topGenes,], cluster_rows=T, scale="row", fontsize=6,fontsize_row=6,fontsize_col=6,show_rownames=T, cluster_cols=T, annotation_col=annot, labels_col=as.character(rownames(df)), main = paste("Heatmap of top 50 DE genes:", contrast[2], "vs", contrast[3]))
dev.off()

# Variance Heatmap
pdf(var_heat)
topVarGenes <- head(order(rowVars(assay(rld)), decreasing = TRUE), 50)
mat  <- assay(rld)[ topVarGenes, ]
mat  <- mat - rowMeans(mat)
pheatmap(mat, scale="row", annotation_col = annot,fontsize=6, main = paste("Heatmap of top 50 most variable genes:", contrast[2], "vs", contrast[3]))
dev.off()

# sort by p-value
res <- res[order(res$padj),]
write.table(as.data.frame(res), file=out_table, quote=FALSE, sep='\t')
R ggplot2 DESeq2 ggrepel BiocParallel PCAtools pheatmap From line 1 of scripts/deseq2_pairwise.R 
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library(DESeq2)
library(dplyr)
library(ggplot2)

# Generate subdata 
counts <- snakemake@input[['counts']]

metadata <- snakemake@params[['samples']]

sampleID <- snakemake@params[['sample_id']]

hist <- snakemake@output[['histogram']]

numGenes <- snakemake@output[['numGenes']]

permList <- snakemake@output[['permList']]

Type <- snakemake@params[['linear_model']]

contrast <- snakemake@params[['contrast']]

baseline <- contrast[[2]]

target <- contrast[[1]]

md <- read.delim(file=metadata, sep = "\t", stringsAsFactors = FALSE)
md <- md[order(md[[sampleID]]),]

# Read in counts table
subdata <- read.table(counts, header=TRUE, row.names=1, sep="\t")
subdata <- subdata[,order(colnames(subdata))]

# Extract only the Types that we want in further analysis & only the PP_ID and Status informative columns
md <- select(md, sampleID, Type)
md <- filter(md, !!as.name(Type) == baseline | !!as.name(Type) == target, !!as.name(sampleID) %in% colnames(subdata))

# Keep only the PP_IDs of the types we have chosen in the metadata table above
rownames(md) <- md[[sampleID]]
md[[sampleID]] <- NULL
keep <- colnames(subdata)[colnames(subdata) %in% rownames(md)]
subdata <- subdata[, keep]
dim(subdata)

# Check
stopifnot(rownames(md)==colnames(subdata))

# Get the number of Cancer samples and number of HD samples from md table
num1 = sum(md[[Type]] == baseline)
num2 = sum(md[[Type]] == target)

# Create a vector for both HD and Can, with a 1 for every HD and a 2 for every Cancer sample
One_vector = rep(c(1), times = num1)
Two_vector = rep(c(2), times = num2)

# Permutation
# Concatenate the HD and Can vector to create your "start group" vector
start_group = c(One_vector, Two_vector)
cutoff=0.01
number_of_diff_genes=c()
group_list = list()
number_of_try = 10

for (i in 1:number_of_try)
{
  print(i)
  group = data.frame(type=factor(sample(start_group)))

  dds = DESeqDataSetFromMatrix(countData = subdata,
                               colData = group,
                               design = ~ type)

  # Extract normalized counts
  dds = estimateSizeFactors(dds)

  # Remove genes with zero counts over all samples
  dds <- dds[ rowSums(counts(dds)) >= 1, ]

  # Make sure of reference, set it by rlevel
  dds$type = relevel(dds$type, ref = 1)

  # The standard differential expression analysis steps are wrapped into a single function, DESeq
  dds = DESeq(dds)

  # Extract results
  res = results(dds, contrast = c("type", "1", "2"), independentFiltering = FALSE,cooksCutoff = Inf)

  tmp=sum(res$padj < cutoff, na.rm=TRUE)
  number_of_diff_genes = c(number_of_diff_genes,tmp)
  group_list[[i]] <- group

}

# Obtain the number of genes that meet padj<0.01 for reference line in histogram
dds <- DESeqDataSetFromMatrix(countData=subdata,
                              colData=md,
                              design= as.formula(paste('~',Type)))

dds <- estimateSizeFactors(dds)

# Remove uninformative columns
dds <- dds[ rowSums(counts(dds)) >= 1, ]

# Normalization and pre-processing
dds <- DESeq(dds)

# Extract results and the number of significant genes with padj<0.01
results = results(dds, contrast = c(Type, target, baseline), independentFiltering = FALSE,cooksCutoff = Inf)
numSig <- sum(results$padj < cutoff, na.rm=TRUE)
number_of_diff_genes <- as.data.frame(number_of_diff_genes)
names(number_of_diff_genes) <- "NumDiffGenes"
number_of_diff_genes$Actual <- numSig

p <- ggplot(number_of_diff_genes, aes(x=NumDiffGenes)) +
  geom_histogram(bins=100) +
  geom_vline(data=number_of_diff_genes, mapping=aes(xintercept = numSig, color = "Correct Labels"), 
             linetype="longdash", size=0.6, show.legend = T) +
  scale_color_manual(values = "gray75", name = "Number of DE genes") +
  ggtitle(paste(number_of_try, "Random Permutations:", baseline, "vs", target)) +
  xlab("Number of significant genes") +
  theme(aspect.ratio=1,
        plot.title = element_text(hjust = 0.5),
        legend.title = element_text(size=10, hjust = 0.5))

pdf(hist)
print({
  p
})
dev.off()

df <- data.frame(stringsAsFactors = FALSE)

for (i in 1:number_of_try) {
  if (i==1) {
    df = group_list[[i]]
  }
  else {
    df = cbind(df, group_list[[i]])
  }
  colnames(df)[i] = paste("perm",i, sep = "_")
}

write.csv(number_of_diff_genes, numGenes)
write.csv(df, permList)
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library("DESeq2")
library("reshape2")
library("cowplot")
library("limma")
library("vsn")
library("genefilter")
library("ggplot2")
library("dplyr")
library("RColorBrewer")
library("pheatmap")
library("hexbin")

# output files
MDS_out <- snakemake@output[['mds_plot']]
MDS_table <- snakemake@output[['mds_table']]
heatmap_out <- snakemake@output[['heatmap_plot']]
sd_out <- snakemake@output[['sd_plot']]
normCounts_out <- snakemake@output[['rlogCounts_plot']]
normCounts_fac <- snakemake@output[['rlogCounts_fac_plot']]
rawCounts_out <- snakemake@output[['counts_plot']]
rawCounts_fac <- snakemake@output[['counts_fac_plot']]

# parameters
sampleID <- snakemake@params[['sample_id']]
Type = snakemake@params[['linear_model']]
plot_cols <- snakemake@config[['meta_columns_to_plot']]
subset_cols = names(plot_cols)

# color palette
colors <- snakemake@params[['colors']]
discrete <- snakemake@params[['discrete']]

# DESeq2 objects
rld <- snakemake@input[['rld']]
dds <- snakemake@input[['rds']]

rld <- readRDS(rld)
dds <- readRDS(dds)

# function to grab the ggplot2 colours
gg_color_hue <- function(n) {
  hues = seq(15, 375, length = n + 1)
  hcl(h = hues, l = 65, c = 100)[1:n]
}

rawCounts <- counts(dds, normalized=FALSE)
md <- as.data.frame(colData(rld))
md$SampleID <- rownames(md)

if(colors[[1]] !='NA' & discrete[[1]] =='NA'){
    if (brewer.pal.info[colors[[1]],]$maxcolors >= length(unique(md[[Type]]))) {
        pal <- brewer.pal(length(unique(md[[Type]])),name=colors[[1]])
    } 
} else if(discrete[[1]] != 'NA' & length(discrete)==length(unique(md[[Type]]))){
        pal <- unlist(discrete)
} else {
        pal <- gg_color_hue(length(unique(md[[Type]])))
}


df1 <- melt(rawCounts) %>%
  dplyr::rename(Gene=Var1) %>%
  dplyr::rename(SampleID=Var2) %>%
  dplyr::rename(counts=value)

iv <- match(df1$SampleID, md$SampleID)
df1$Condition <- paste(md[iv,][[Type]])
df1$SampleID <- factor(df1$SampleID, levels=unique(md$SampleID))

# aesthetic for plots
dodge <- position_dodge(width = 0.6)
theme_update(plot.title = element_text(hjust = 0.5))

p1 <- ggplot(data=df1, mapping=aes(x=SampleID, y=counts, fill=Condition)) +
  geom_violin(width=0.7) +
  geom_boxplot(width=0.2, outlier.colour=NA, position = dodge, color="gray28") +
  scale_y_log10() +
  scale_fill_manual(values=pal) +
  theme(axis.text.x = element_text(hjust=1, angle=45, size=6))

# width of pdf to ensure all sampleIDs are visible when exported to pdf
# This was generated with a use case of 16 samples and a width of 7 fitting well, the +8 is to account for the margins
width <- 7/24*(nrow(md)+8)

# raw counts boxplot
pdf(rawCounts_out, width, 5)
print({
  p1
})
dev.off()

# faceted by condition
p2 <- ggplot(data=df1, mapping=aes(x=SampleID, y=counts, fill=Condition)) +
  geom_violin(width=0.7) +
  geom_boxplot(width=0.2, outlier.colour=NA, position = dodge, color="gray28") +
  scale_y_log10() +
  scale_fill_manual(values=pal) +
  theme(axis.text.x = element_text(hjust=1, angle=45, size=4)) +
  facet_wrap(~Condition)

pdf(rawCounts_fac, 2*width, 5)
print({
  plot_grid(p1, p2)
})
dev.off()

# Run same analysis for log2-transformed normalized counts
df2 <- melt(assay(rld)) %>%
  dplyr::rename(Gene=Var1) %>%
  dplyr::rename(SampleID=Var2) %>%
  dplyr::rename(normCounts=value)

# Add Condition information to this dataframe
iv <- match(df2$SampleID, md$SampleID)
df2$Condition <- paste(md[iv,][[Type]])
df2$SampleID <- factor(df2$SampleID, levels=unique(md$SampleID))

p1 <- ggplot(data=df2, mapping=aes(x=SampleID, y=normCounts, fill=Condition)) +
  geom_violin(width=0.7) +
  geom_boxplot(width=0.2, outlier.colour=NA, position = dodge, color="gray28") +
  scale_fill_manual(values=pal) +
  theme(axis.text.x = element_text(hjust=1, angle=45, size=6)) +
  ylab("regularized log expression")

# raw counts boxplot
pdf(normCounts_out, width, 5)
print({
  p1
})
dev.off()

# faceted by condition
p2 <- ggplot(data=df2, mapping=aes(x=SampleID, y=normCounts, fill=Condition)) +
  geom_violin(width=0.7) +
  geom_boxplot(width=0.2, outlier.colour=NA, position = dodge, color="gray28") +
  scale_fill_manual(values=pal) +
  theme(axis.text.x = element_text(hjust=1, angle=45, size=4)) +
  facet_wrap(~Condition) +
  ylab("regularized log expression")

pdf(normCounts_fac, 2*width, 5)
print({
  plot_grid(p1, p2)
})
dev.off()

# Standard deviation vs. mean
ntd <- normTransform(dds)

pdf(sd_out)
meanSdPlot(assay(ntd))
dev.off()

# Generate annotation column for heatmap
if (length(subset_cols)==1) {
  annot <- as.data.frame(cbind(rownames(md), paste(md[[subset_cols[1]]])))
  names(annot) <- c("SampleID", subset_cols[1])
  rownames(annot) <- annot$SampleID
  annot$SampleID <- NULL
} else {
  annot <- md[,subset_cols]
}

hm <- pheatmap(assay(rld), show_rownames=F, clustering_distance_rows = "correlation", 
         clustering_distance_cols = "correlation", clustering_method = "average", 
         annotation_col = annot, scale = "row", 
         main="Unsupervised heatmap of all gene counts across samples",
         fontsize_row=4, fontsize_col=6, fontsize=8,
         color = colorRampPalette(c("navy", "white", "firebrick3"))(50))

save_pheatmap_pdf <- function(x, filename) {
  stopifnot(!missing(x))
  stopifnot(!missing(filename))
  pdf(filename)
  grid::grid.newpage()
  grid::grid.draw(x$gtable)
  dev.off()
}

save_pheatmap_pdf(hm, heatmap_out)

# use plotMA function from limma, then extract data from this variable to plot with ggplot2
p <- plotMDS(assay(rld), top = 1000)
df <- data.frame(x=p$x, y=p$y, name=names(p$x))
iv <- match(df$name, md$SampleID)
df$Condition <- paste(md[iv,][[Type]])

pdf(MDS_out)
ggplot(data=df, mapping=aes(x=x,y=y)) +
  geom_point(size=3, colour = "black", show.legend = TRUE) +
  geom_point(aes(color=Condition), size=2.2) +
  scale_colour_manual(values=pal) +
  xlab("Leading logFC dim 1") +
  ylab("Leading logFC dim 2") +
  ggtitle("MDS Plot")
dev.off()

# Export the table for MDS
write.table(df, file=MDS_table, sep="\t", quote=F, row.names=FALSE)
R ggplot2 dplyr DESeq2 cowplot RColorBrewer reshape2 pheatmap limma genefilter vsn hexbin From line 1 of scripts/QC.R 
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library("data.table")
library("qvalue")

stats_table <- snakemake@input[['stats_table']]
cat(sprintf(c('stats table: ', stats_table, '\n')))


qplot <- snakemake@output[['qplot']]
cat(sprintf(c('Qvalue Output: ', qplot, '\n')))

qhist <- snakemake@output[['qhist']]
cat(sprintf(c('Qvalue hist Output: ', qhist, '\n')))

out_table = snakemake@output[['table']]
cat(sprintf(c('Summary results table', out_table,'\n')))

stats_frame = read.table(stats_table, row.names=1, sep='\t', check.names=F)

qobj = qvalue(p=stats_frame$pvalue, fdr.level=T)

stats_frame$qvalues = qobj$qvalues
stats_frame$lfdr = qobj$lfdr
write.table(as.data.frame(stats_frame), file=out_table, quote=FALSE, sep='\t')

pdf(qplot)
plot(qobj)
dev.off()

pdf(qhist)
hist(qobj)
dev.off()
R data.table qvalue From line 1 of scripts/qplot.R 
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annoFile = snakemake@params[['anno']]

biotypes <- snakemake@params[['biotypes']]

countsFile <- snakemake@input[['countsFile']]

mito <- snakemake@params[['mito']]

counts <- read.delim(file=countsFile)

##----------load counts------------#
print("Loading counts table")
print(countsFile)

## must be a tsv or txt tab sep file

counts <- read.delim(file=countsFile)

##----------load anno------------#
print("Loading annotation table")
print(annoFile)

## load
anno <- get(load(file=annoFile))

if(strsplit(biotypes, split='\\,')[[1]]!=""){
    anno.sub <- anno[paste(anno$gene_biotype) %in% strsplit(biotypes, split='\\,')[[1]] ,]
    counts.sub <- counts[paste(counts$Genes) %in% unique(paste(anno.sub$external_gene_name)) , ]
}else{
    print("no biotypes provided")
    counts.sub <- counts
}

if(mito==1){
    print("tossing MT- genes")
    counts.sub <- counts.sub[grep("^MT-", paste(counts.sub$Genes), invert=TRUE), ]
}

write.table(counts.sub, file=sub(".txt", ".filt.txt", countsFile), sep="\t", quote=FALSE, row.names=FALSE, col.names=TRUE)
R From line 1 of scripts/RNAseq_filterCounts.R 
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library(ggplot2)
library(ggrepel)

degFile = snakemake@input[['degFile']]

FC <- snakemake@params[['FC']]

adjp <- snakemake@params[['adjp']]

contrast <- snakemake@params[['contrast']]

baseline <- contrast[[2]]

target <- contrast[[1]]

volcano_plot=snakemake@output[['volcano_plot']]

upCol = "#FF9999"
downCol = "#99CCFF"
ncCol = "#CCCCCC"

##----------load differentially expressed genes --------#
print("Loading differential expressed gene table")
print(degFile)

## check if an rda file or tab sep
deg <- read.delim(file=degFile)

head(deg)
dim(deg)

## set all NA missing p-values to 1 (NA is DESeq2 default)
deg[is.na(deg$padj), "padj"] <- 1

## select up regulated genes
up <- deg$padj < adjp & deg$log2FoldChange > log2(FC)
sum(up)

## select down regulated genes
down <- deg$padj < adjp & deg$log2FoldChange < -log2(FC)
sum(down)

# Grab the top 5 up and down regulated genes to label in the volcano plot
if (sum(up)>5) {
  upGenesToLabel <- head(rownames(deg[up,]), 5)
} else if (sum(up) %in% 1:5) {
  upGenesToLabel <- rownames(deg[up,])
}

if (sum(down)>5) {
  downGenesToLabel <- head(rownames(deg[down,]), 5)
} else if (sum(down) %in% 1:5) {
  downGenesToLabel <- rownames(deg[down,])
}

## calculate the -log10(adjp) for the plot
deg$log10padj <- -log10(deg$padj)

# assign up and downregulated genes to a category so that they can be labeled in the plot
deg$Expression <- ifelse(down, 'down',
                  ifelse(up, 'up','NS'))
deg$Expression <- factor(deg$Expression, levels=c("up","down","NS"))

# Assign colours to conditions
if (sum(up)==0 & sum(down)==0) {
  colours <- ncCol
} else if (sum(up)==0) {
  colours <- c(downCol, ncCol)
} else if (sum(down)==0) {
  colours <- c(upCol, ncCol)
} else {
  colours <- c(upCol, downCol, ncCol)
}

# Set all Infinity values to max out at 500 so that all points are contained in the plot
if ("Inf" %in% deg$log10padj) {
  deg$log10padj[deg$log10padj=="Inf"] <- max(deg[is.finite(deg$log10padj),"log10padj"]) + 2
}

deg$Gene <- rownames(deg)

# Assign genes to label based on whether genes are DE or not
if (exists("downGenesToLabel") & exists("upGenesToLabel")) {
  genesToLabel <- c(downGenesToLabel, upGenesToLabel)
} else if (exists("downGenesToLabel") & !exists("upGenesToLabel")) {
  genesToLabel <- downGenesToLabel
} else if (!exists("downGenesToLabel") & exists("upGenesToLabel")) {
  genesToLabel <- upGenesToLabel
}

if (exists("genesToLabel")) {
  p <- ggplot(data=deg, mapping=aes(x=log2FoldChange, y=log10padj, colour=Expression)) +
    geom_vline(xintercept = c(-log2(FC),log2(FC)), linetype="dashed", colour="gray45") +
    geom_hline(yintercept = -log10(adjp), linetype="dashed", colour="gray45") +
    geom_label_repel(aes(label=ifelse(Gene %in% genesToLabel, as.character(Gene),'')),box.padding=0.1, point.padding=0.5, segment.color="gray70", show.legend=FALSE) +
    geom_point() +
    ylab("-log10(FDR)") +
    xlab("log2(Fold Change)") +
    ggtitle(paste(target, "vs", baseline)) +
    scale_colour_manual(values=colours) +
    theme(plot.title = element_text(hjust = 0.5, face="plain"),
          axis.title.x = element_text(size=11),
          axis.title.y = element_text(size=11),
          panel.background = element_blank(),
          axis.line = element_line(colour = "gray45"),
          legend.key = element_rect(fill = "gray96"),
          legend.text = element_text(size = 10))
} else {
  p <- ggplot(data=deg, mapping=aes(x=log2FoldChange, y=log10padj, colour=Expression)) +
    geom_vline(xintercept = c(-log2(FC),log2(FC)), linetype="dashed", colour="gray45") +
    geom_hline(yintercept = -log10(adjp), linetype="dashed", colour="gray45") +
    geom_point() +
    geom_vline(xintercept = c(-log2(FC),log2(FC)), linetype="dashed", colour="gray45") +
    geom_hline(yintercept = -log10(adjp), linetype="dashed", colour="gray45") +
    ylab("-log10(FDR)") +
    xlab("log2(Fold Change)") +
    ggtitle(paste(target, "vs", baseline)) +
    scale_colour_manual(values=colours) +
    theme(plot.title = element_text(hjust = 0.5, face="plain"),
          axis.title.x = element_text(size=11),
          axis.title.y = element_text(size=11),
          panel.background = element_blank(),
          axis.line = element_line(colour = "gray45"),
          legend.key = element_rect(fill = "gray96"),
          legend.text = element_text(size = 10))
}


pdf(volcano_plot)
print({
  p
})
dev.off()
R ggplot2 DESeq2 ggrepel From line 1 of scripts/RNAseq_makeVolcano.R 
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library(Glimma)
library(limma)
library(DESeq2)

project_id = snakemake@params[['project_id']]

rds = snakemake@input[['rds']]
cat(sprintf(c('RDS object: ',rds,'\n')))


out_path = file.path(getwd(),'results','diffexp')
dir.create(out_path)

rds = readRDS(rds)
groups.df = as.data.frame(colData(rds))
glMDSPlot(rds, groups=groups.df,path=out_path,html=paste(project_id,'mds_plot',sep='.'),launch=FALSE)
R Glimma From line 1 of scripts/run_glimma_mds.R 
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library(Glimma)
library(limma)
library(DESeq2)

condition = snakemake@params[['condition']]
cat(sprintf(c('Condition: ',condition,'\n')))

title = snakemake@params[["contrast"]]
print(title)
contrast = c(condition, snakemake@params[["contrast"]])
rds = snakemake@input[['rds']]
cat(sprintf(c('RDS object: ',rds,'\n')))

md_path = snakemake@output[['ma_plot']]
va_path = snakemake@output[['volcano_plot']]
mdout = tail(strsplit(md_path,'/')[[1]],n=1)
mdout = strsplit(mdout,'.html')[1]
vaout = tail(strsplit(va_path, '/')[[1]],n=1)
vaout = strsplit(vaout,'.html')[1]

out_path = file.path(getwd(),'results','diffexp')
dir.create(out_path)
print(out_path)
rds = readRDS(rds)
groups.df = as.data.frame(colData(rds))


#### by contrasts
#contrasts_to_plot = resultsNames(rds)
res <- results(rds, contrast=contrast)
res$padj[is.na(res$padj)] = 1

rnaseq = as.data.frame(counts(rds, normalized=T))
genes = as.data.frame(row.names(res))
colnames(genes) = 'GeneID'

status_frame = res[,c('log2FoldChange','padj')]
status_frame['status'] = 0
status_frame$padj[is.na(status_frame$padj)] = 1
status_frame[status_frame$padj<0.05 & status_frame$log2FoldChange < 0 ,'status'] = -1
status_frame[status_frame$padj<0.05 & status_frame$log2FoldChange > 0 ,'status'] = 1

title = paste(title[1],'vs',title[2],sep='-')

glMDPlot(res, anno=genes, status=status_frame$status, samples=colnames(rnaseq), 
         counts=log2(rnaseq + 0.0001), groups=groups.df[[condition]], main=strsplit(res@elementMetadata$description[2],': ')[[1]][2], 
         transform=F, side.ylab='Log2-expression',launch=FALSE,side.main='GeneID', html = mdout, path=out_path)

## Volcano plot
glXYPlot(x=res$log2FoldChange, y=-log10(res$pvalue), xlab="logFC", ylab="logodds",path=out_path,
         status=status_frame$status, launch=FALSE,counts=log2(rnaseq + 0.0001), groups=groups.df[[condition]], anno=genes,main=strsplit(res@elementMetadata$description[2],': ')[[1]][2], html = vaout)
R Glimma From line 1 of scripts/run_glimma.R 
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degFile = snakemake@input[['degFile']]

assembly <- snakemake@params[['assembly']]

FC <- snakemake@params[['FC']]

adjp <- snakemake@params[['adjp']]

printTree <- snakemake@params[['printTree']]

library(GO.db)
library(topGO)
library(ggplot2)
library(RColorBrewer)
library(biomaRt)
library(GenomicFeatures)
library(Rgraphviz)

##----------load differentially expressed genes --------#
print("Loading differential expressed gene table")
print(degFile)

if(grepl('txt$|tsv$',degFile)){
    deg <- read.delim(file=degFile,header=TRUE,sep="\t")
}

##---------load correct Biomart------------------------#
print(getwd())
if (assembly == "hg19") {
    organismStr <- "hsapiens"
    geneID2GO <- get(load("./anno/biomaRt/hg19.Ens_75.biomaRt.GO.external.geneID2GO.RData"))
    xx <- get(load("./anno/biomaRt/GO.db.Term.list.rda"))
}
if (assembly == "hg38.89") {
    organismStr <- "hsapiens"
    ### to get to hg38 mappings ensembl 89!
    geneID2GO <- get(load("./anno/biomaRt/hg38.Ens_89.biomaRt.GO.external.geneID2GO.RData"))
    xx <- get(load("./anno/biomaRt/GO.db.Term.list.rda"))
}
if (assembly == "hg38.90") {
    organismStr <- "hsapiens"
    ### to get to hg38 mappings ensembl 90!
    geneID2GO <- get(load("./anno/biomaRt/hg38.Ens_90.biomaRt.GO.external.geneID2GO.RData"))
    xx <- get(load("./anno/biomaRt/GO.db.Term.list.rda"))
}
if (assembly == "mm10.78") {
    organismStr <- "mmusculus"
    ### to get to hg38 mappings ensembl 90!
    geneID2GO <- get(load("./anno/biomaRt/mm10.Ens_78.biomaRt.GO.external.geneID2GO.RData"))
    xx <- get(load("./anno/biomaRt/GO.db.Term.list.rda"))
}
if (assembly == "mm10.96") {
    organismStr <- "mmusculus"
    ### to get to hg38 mappings ensembl 90!
    geneID2GO <- get(load("./anno/biomaRt/mm10.Ens_96.biomaRt.GO.external.geneID2GO.RData"))
    xx <- get(load("./anno/biomaRt/GO.db.Term.list.rda"))
}


##-----------------------------------Functions--------------------------------------#
runGO <- function(geneList,xx=xx,otype,setName){
    setLength       <- sum(as.numeric(levels(geneList))[geneList]) 
    fname           <- paste(Dir, paste(setName, otype, "GO.txt", sep="_"), sep="/")
    GOData          <- new("topGOdata", ontology=otype, allGenes=geneList, annot = annFUN.gene2GO, gene2GO = geneID2GO)
    resultFisher    <- runTest(GOData, algorithm = "classic", statistic = "fisher")## statistical test for topGO
    x               <- GenTable(GOData, classicFisher=resultFisher, topNodes=length(names(resultFisher@score)))## make go table for all terms
    x               <- data.frame(x)
    pVal            <- data.frame(pval=signif(resultFisher@score, 6)) ## get unrounded pvalue
    x$enrich        <- x$Significant/x$Expected ## calculate enrichment based on what you expect by chance
    x$p.unround     <- pVal[x$GO.ID,"pval"]## put unrounded pvalue in the table
    x$p.adj         <- signif(p.adjust(x$p.unround, method="BH"), 6)## calculate the adjusted pvalue with Benjamini & Hochberg correction
    x$log.p.adj     <- -log10(x$p.adj) ## convert adjusted p value to -log10 for plot magnitude
    #x$Term.full     <- sapply(x$GO.ID, FUN=function(n){Term(xx[[n]])}) ## get the full term name
    x <- x[order(x$GO.ID),]
    write.table(x, file=fname, sep="\t", col.names=TRUE, quote=FALSE, row.names=FALSE) ## save the table
    ## you can print the tree if you want, but since I keep the list of all of them skip
    if(printTree>0){
        printGraph(GOData,## make the tree for the go data
                   resultFisher,
                   firstSigNodes = 5,
                   fn.prefix = sub("_GO.txt$", "", fname),
                   useInfo = "all",
                   pdfSW = TRUE
                   )
    }
    return(x)  
}

## function to make barplot of -log10 adjusted pvalues colored by enrichment

drawBarplot <- function(go, ontology, setName){
    go <- go[!go$p.adj > 0.01,]
    if(nrow(go)>1){
        #go$Term.full <- make.unique(paste(sapply(strsplit(as.character(substring(go$Term.full,1,50)), "\\,"), `[`, 1)))
        go$Term <- make.unique(paste(sapply(strsplit(as.character(substring(go$Term,1,50)), "\\,"), `[`, 1)))
        print(setName)
        go <- go[with(go, order(p.adj, -enrich)),]
        ## Currently there is a discrepency between xx and x, so we only use Term right now, not Term.full
        #go$Term.full <-factor(paste(go$Term.full), levels=rev(paste(go$Term.full))) ## sort table by adjusted p-value
        go$Term <-factor(paste(go$Term), levels=rev(paste(go$Term))) ## sort table by adjusted p-value
        ptitle <- paste(ontology, setName) ## plot title
        ptitle <- gsub("^.*/","",ptitle)
        pfname <- paste(setName,ontology,"pdf",sep=".")## name of png file
        if(nrow(go) < 20 ){
            toprange <- 1:nrow(go)
        }else{
            toprange <- 1:20
        }
        top <- go[toprange,]
        col <- colorRampPalette(c("white","navy"))(16)   
        pdf(file=paste(Dir, pfname, sep="/"),height=5,width=7)
        print({
           p <- ggplot(top, aes(y=log.p.adj, x=Term, fill=enrich)) + ## ggplot barplot function
               geom_bar(stat="identity",colour="black") +
               ggtitle(ptitle) +
               xlab("") + ylab("-log10(fdr)") +
               scale_fill_gradient(low=col[2], high=col[15], name="enrichment", limits=c(0,ceiling(max(top$enrich))))+
               coord_flip()+
               theme(panel.grid.major = element_line(colour = "grey"), panel.grid.minor = element_blank(), 
                     panel.background = element_blank(), axis.line = element_line(colour = "black"))+
               theme(text = element_text(size=8),
                     axis.text.x = element_text(vjust=1,color="black",size=8),
                     axis.text.y = element_text(color="black",size=8),
                     plot.title=element_text(size=10))   
       })
       dev.off()
    }
}

print("get up genes and make geneList")
up <- deg$padj < adjp & deg$log2FoldChange >= log2(FC)
up <- unique(rownames(deg[up,]))
up <- toupper(up)
all <-unique(names(geneID2GO))
up.geneList <-  factor(as.integer(all %in% up))
names(up.geneList) <- all

up.setsize <- sum(as.numeric(levels(up.geneList))[up.geneList])
print("setsize for significant genes") 
up.setsize

adjplabel <- gsub("^0\\.","",adjp)
comparison <- gsub("\\.tsv$|\\.txt$|\\.rda$|\\.RData$","",degFile)

Dir <- sub("$", "/GOterms", dirname(comparison))
if(!(file.exists(Dir))) {
      dir.create(Dir,FALSE,TRUE)
}


if (up.setsize >= 2){

print("make GO table for the up genes")
go.UP.BP <- runGO(geneList=up.geneList,xx=xx,otype="BP",setName=paste(basename(comparison),"upFC",FC, "adjp", adjp, sep="."))
drawBarplot(go=go.UP.BP,ontology="BP",setName=paste(basename(comparison),"upFC",FC, "adjp", adjp, sep="."))

}else{
up_out = snakemake@output[[1]]
write.table('No Significant Genes', file=up_out)
}

print("get down genes and make geneList")
dn <- deg$padj < adjp & deg$log2FoldChange <= -log2(FC)
dn <- unique(rownames(deg[dn,]))
dn <- toupper(dn)
all <-unique(names(geneID2GO))
dn.geneList <-  factor(as.integer(all %in% dn))
names(dn.geneList) <- all

dn.setsize <- sum(as.numeric(levels(dn.geneList))[dn.geneList])
print("setsize for significant genes") 
dn.setsize

if(dn.setsize >= 2){

print("make GO table for down genes")
go.DN.BP <- runGO(geneList=dn.geneList,xx=xx,otype="BP",setName=paste(basename(comparison),"downFC",FC, "adjp", adjp, sep="."))
print("make barplot for down genes")
drawBarplot(go=go.DN.BP,ontology="BP",setName=paste(basename(comparison),"downFC",FC, "adjp", adjp, sep="."))

}else{
down_out = snakemake@output[[2]]
write.table('No Significant Genes', file=down_out)
}
R ggplot2 RColorBrewer GO.db biomaRt Rgraphviz topGO From line 1 of scripts/runGOforDESeq2.R
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/ohsu-cedar-comp-hub/Bulk-RNA-seq-pipeline-SE
Name: bulk-rna-seq-pipeline-se
Version: 2
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