Add new rules visualizing CARLISLE results

CHANGELOG.md

@@ -1,16 +1,19 @@
 ## CARLISLE development version
 
-- Bug fixes (#127, @epehrsson)
+- Bug fixes: (#127, @epehrsson)
     - Removes single-sample group check for DESeq.
     - Increases memory for DESeq.
     - Ensures control replicate number is an integer.
     - Fixes FDR cutoff misassigned to log2FC cutoff.
     - Fixes `no_dedup` variable names in library normalization scripts.
 - Containerize rules that require R (`deseq`, `go_enrichment`, and `spikein_assessment`) to fix installation issues with common R library path. (#129, @kelly-sovacool)
     - The `Rlib_dir` and `Rpkg_config` config options have been removed as they are no longer needed.
+- New visualizations: (#132, @epehrsson)
+    - New rules `cov_correlation`, `homer_enrich`, `combine_homer`, `count_peaks`
+    - Add peak caller to MACS2 peak xls filename
 - New parameters in the config file to make certain rules optional: (#133, @kelly-sovacool)
     - GO enrichment is controlled by `run_go_enrichment` (default: `false`)
-    - rose is controlled by `run_rose` (default: `false`)
+    - ROSE is controlled by `run_rose` (default: `false`)
 - Fig bug that added nonexistent directories to the singularity bind paths. (#135, @kelly-sovacool)
 
 ## CARLISLE v2.5.0

config/config.yaml

@@ -164,4 +164,4 @@ adapters: "PIPELINE_HOME/resources/other/adapters.fa"
 #####################################################################################
 containers:
   base: "docker://nciccbr/ccbr_ubuntu_base_20.04:v6"
-  carlisle_r: "docker://nciccbr/carlisle_r:v1"
+  carlisle_r: "docker://nciccbr/carlisle_r:v2"

docker/carlisle_r/Dockerfile

@@ -9,13 +9,13 @@ ARG REPONAME="000000"
 ENV REPONAME=${REPONAME}
 
 # install conda packages
-COPY packages.txt /data2/
-RUN mamba install \
-    --no-channel-priority \
-    -c bioconda -c conda-forge -c r \
-    --file /data2/packages.txt
-ENV PATH="/opt2/conda/bin:$PATH"
+COPY environment.yml /data2/
+ENV CONDA_ENV=carlisle
+RUN mamba env create -n ${CONDA_ENV} -f /data2/environment.yml && \
+    echo "conda activate ${CONDA_ENV}" > ~/.bashrc
+ENV PATH="/opt2/conda/envs/${CONDA_ENV}/bin:$PATH"
 ENV R_LIBS_USER=/opt2/conda/lib/R/library/
+
 # install ELBOW manually, fails with mamba
 RUN wget --no-check-certificate https://bioconductor.riken.jp/packages/3.4/bioc/src/contrib/ELBOW_1.10.0.tar.gz && \
     R -e 'install.packages("ELBOW_1.10.0.tar.gz", repos = NULL, type="source", INSTALL_opts = "--no-lock")'

docker/carlisle_r/environment.yml

@@ -0,0 +1,38 @@
+channels:
+  - bioconda
+  - conda-forge
+  - r
+dependencies:
+  - bioconductor-bsgenome.hsapiens.ncbi.t2t.chm13v2.0
+  - bioconductor-chipenrich
+  - bioconductor-chipseeker
+  - bioconductor-ComplexHeatmap
+  - bioconductor-deseq2
+  - bioconductor-edger
+  - bioconductor-enhancedvolcano
+  - bioconductor-genomicfeatures
+  - bioconductor-htsfilter
+  - bioconductor-org.Hs.eg.db
+  - bioconductor-org.Mm.eg.db
+  - bioconductor-rtracklayer
+  - bioconductor-txdb.hsapiens.ucsc.hg19.knowngene
+  - bioconductor-TxDb.Hsapiens.UCSC.hg38.knownGene
+  - bioconductor-TxDb.Mmusculus.UCSC.mm10.knownGene
+  - deeptools
+  - r-argparse
+  - r-circlize
+  - r-DT
+  - r-ggfortify
+  - r-ggvenn
+  - r-htmltools
+  - r-latticeextra
+  - r-openxlsx
+  - r-pander
+  - r-pdp
+  - r-plotly
+  - r-plyr
+  - r-rcolorbrewer
+  - r-reshape2
+  - r-tidyverse
+  - r-xfun>=0.43
+  - r-yaml

docker/carlisle_r/meta.yml

@@ -1,4 +1,4 @@
 dockerhub_namespace: nciccbr
 image_name: carlisle_r
-version: v1
+version: v2
 container: "$(dockerhub_namespace)/$(image_name):$(version)"

resources/cluster_biowulf.yaml

@@ -78,3 +78,5 @@ DESeq:
     time: 00-02:00:00
     threads: 2
 ###################################################################
+cov_correlation:
+    threads: 4

workflow/Snakefile

@@ -144,6 +144,26 @@ def get_motifs(wildcards):
         files.extend(anno_s)
     return files
 
+def get_enrich(wildcards):
+    files=[]
+    if ("macs2_narrow" in PEAKTYPE) or ("macs2_broad" in PEAKTYPE):
+        anno_m=expand(join(RESULTSDIR,"peaks","{qthresholds}","{peak_caller}","annotation","homer","enrichment.{dupstatus}.{peak_caller_type}.png"),peak_caller="macs2",qthresholds=QTRESHOLDS,dupstatus=DUPSTATUS,peak_caller_type=PEAKTYPE_M),
+        files.extend(anno_m)
+    if ("gopeaks_narrow" in PEAKTYPE) or ("gopeaks_broad" in PEAKTYPE):
+        anno_g=expand(join(RESULTSDIR,"peaks","{qthresholds}","{peak_caller}","annotation","homer","enrichment.{dupstatus}.{peak_caller_type}.png"),peak_caller="gopeaks",qthresholds=QTRESHOLDS,dupstatus=DUPSTATUS,peak_caller_type=PEAKTYPE_G),
+        files.extend(anno_g)
+    if ("seacr_stringent" in PEAKTYPE) or ("seacr_relaxed" in PEAKTYPE):
+        anno_s=expand(join(RESULTSDIR,"peaks","{qthresholds}","{peak_caller}","annotation","homer","enrichment.{dupstatus}.{peak_caller_type}.png"),peak_caller="seacr",qthresholds=QTRESHOLDS,dupstatus=DUPSTATUS,peak_caller_type=PEAKTYPE_S),
+        files.extend(anno_s)
+    return files
+
+def get_combined(wildcards):
+    files = []
+    if ("macs2_narrow" in PEAKTYPE) or ("macs2_broad" in PEAKTYPE):    
+        combined_m = expand(join(RESULTSDIR,"peaks","{qthresholds}","macs2","annotation","homer","{treatment_control_list}.{dupstatus}.{peak_caller_type}.annotation_qvalue.tsv"),qthresholds=QTRESHOLDS,dupstatus=DUPSTATUS,peak_caller_type=PEAKTYPE_M,treatment_control_list=TREATMENT_LIST_M)
+        files.extend(combined_m)
+    return files
+
 def get_rose(wildcards):
     files=[]
     if config['run_rose']:
@@ -200,6 +220,12 @@ rule all:
         # ALIGN / deeptools_heatmap
         unpack(run_deeptools_heatmap),
 
+        # ALIGN / deeptools_corr
+        expand(join(RESULTSDIR,"deeptools","all.{dupstatus}.PearsonCorr.tab"),dupstatus=DUPSTATUS),
+        expand(join(RESULTSDIR,"deeptools","all.{dupstatus}.PCA.tab"),dupstatus=DUPSTATUS),
+        expand(join(RESULTSDIR,"deeptools","all.{dupstatus}.Pearson_heatmap.png"),dupstatus=DUPSTATUS),
+        expand(join(RESULTSDIR,"deeptools","all.{dupstatus}.PearsonPCA.png"),dupstatus=DUPSTATUS),
+
         # ALIGN / create_library_norm_scales
         unpack(run_library_norm),
 
@@ -213,6 +239,8 @@ rule all:
         unpack(run_macs2),
         unpack(run_seacr),
         unpack(run_gopeaks),
+        join(RESULTSDIR,"peaks","Peak counts.xlsx"),
+        join(RESULTSDIR,"peaks","all.peaks.txt"),
 
         # QC
         unpack(run_qc),
@@ -222,6 +250,8 @@ rule all:
 
         # ANNOTATION / findMotif, rose, create_contrast_peakcaller_files, go_enrichment
         unpack(get_motifs),
+        unpack(get_enrich),
+        unpack(get_combined),
         unpack(get_rose),
         unpack(get_enrichment)
 
@@ -292,4 +322,4 @@ onerror:
         expand(join(RESULTSDIR,"deeptools","clean", "{group}.{dupstatus}.metagene.mat.gz"),group=[a+b for a in TREATMENTS+["all_samples"] for b in ["", ".prot"]],dupstatus=DUPSTATUS),
         expand(join(RESULTSDIR,"deeptools","clean", "{group}.{dupstatus}.TSS.mat.gz"),group=[a+b for a in TREATMENTS+["all_samples"] for b in ["", ".prot"]],dupstatus=DUPSTATUS),
         expand(join(RESULTSDIR,"deeptools","clean", "{group}.{dupstatus}.geneinfo.bed"),group=[a+b for a in TREATMENTS+["all_samples"] for b in ["", ".prot"]],dupstatus=DUPSTATUS),
-"""
+"""

workflow/rules/align.smk

@@ -468,4 +468,48 @@ rule deeptools_heatmap:
         plotHeatmap -m {input.TSSmat} -out {output.TSSheat} --colorMap 'RdBu_r' --zMin auto --zMax auto --yAxisLabel 'average RPGC' --regionsLabel 'genes' --legendLocation 'none'
         plotProfile -m {input.metamat} -out {output.metaline} --plotHeight 15 --plotWidth 15 --perGroup --yAxisLabel 'average RPGC' --plotType 'se' --legendLocation upper-right
         plotProfile -m {input.TSSmat} -out {output.TSSline} --plotHeight 15 --plotWidth 15 --perGroup --yAxisLabel 'average RPGC' --plotType 'se' --legendLocation upper-left
-        """
+        """
+
+rule cov_correlation:
+    """
+    Create replicate correlation plots from filtered BAM files
+    """
+    input:
+        bams=expand(join(RESULTSDIR,"bam","{replicate}.{{dupstatus}}.bam"),replicate=REPLICATES),
+        align_table=join(RESULTSDIR,"alignment_stats","alignment_stats.tsv")
+    output:
+        counts=join(RESULTSDIR,"deeptools","all.{dupstatus}.readCounts.npz"),
+        pearson_corr=join(RESULTSDIR,"deeptools","all.{dupstatus}.PearsonCorr.tab"),
+        pearson_plot=join(RESULTSDIR,"deeptools","all.{dupstatus}.PearsonCorr.png"),
+        pca=join(RESULTSDIR,"deeptools","all.{dupstatus}.PCA.tab"),
+        hc=join(RESULTSDIR,"deeptools","all.{dupstatus}.Pearson_heatmap.png"),
+        pca_format=join(RESULTSDIR,"deeptools","all.{dupstatus}.PearsonPCA.png")        
+    params:
+        rscript=join(SCRIPTSDIR,"_plot_correlation.R"),
+        dupstatus="{dupstatus}"
+    container: config['containers']['carlisle_r']
+    threads: getthreads("cov_correlation")
+    shell:
+        """
+        # Calculate genome-wide coverage
+        multiBamSummary bins \
+         --bamfiles {input.bams} \
+         --smartLabels \
+         -out {output.counts} \
+         -p {threads}
+
+        # Plot heatmap - Pearson
+        plotCorrelation \
+         -in {output.counts} \
+         --corMethod pearson --skipZeros \
+         --whatToPlot heatmap --plotNumbers \
+         -o {output.pearson_plot} \
+         --removeOutliers \
+         --outFileCorMatrix {output.pearson_corr}
+
+        # Plot PCA
+        plotPCA -in {output.counts}  --outFileNameData {output.pca}
+
+        # Plot heatmap and PCA (formatted)
+        Rscript {params.rscript} {output.pearson_corr} {output.pca} {input.align_table} {params.dupstatus} {output.hc} {output.pca_format}
+        """