## ----style, echo = FALSE, results = 'asis'------------------------------- BiocStyle::markdown() options(width=100, max.print=1000) knitr::opts_chunk$set( eval=as.logical(Sys.getenv("KNITR_EVAL", "TRUE")), cache=as.logical(Sys.getenv("KNITR_CACHE", "TRUE"))) ## ----setup, echo=FALSE, messages=FALSE, warnings=FALSE------------------- suppressPackageStartupMessages({ library(systemPipeR) library(BiocParallel) library(Biostrings) library(Rsamtools) library(GenomicRanges) library(ggplot2) library(GenomicAlignments) library(ShortRead) library(ape) }) ## ----install, eval=FALSE------------------------------------------------- ## source("http://bioconductor.org/biocLite.R") # Sources the biocLite.R installation script ## biocLite("systemPipeR") # Installs systemPipeR ## biocLite("systemPipeRdata") # Installs systemPipeRdata ## ----documentation, eval=FALSE------------------------------------------- ## library("systemPipeR") # Loads the package ## library(help="systemPipeR") # Lists package info ## vignette("systemPipeR") # Opens vignette ## ----genRna_workflow, eval=FALSE----------------------------------------- ## library(systemPipeRdata) ## genWorkenvir(workflow="rnaseq") ## setwd("rnaseq") ## ----targetsSE, eval=TRUE------------------------------------------------ library(systemPipeR) targetspath <- system.file("extdata", "targets.txt", package="systemPipeR") read.delim(targetspath, comment.char = "#") ## ----targetsPE, eval=TRUE------------------------------------------------ targetspath <- system.file("extdata", "targetsPE.txt", package="systemPipeR") read.delim(targetspath, comment.char = "#")[1:2,1:6] ## ----comment_lines, eval=TRUE-------------------------------------------- readLines(targetspath)[1:4] ## ----targetscomp, eval=TRUE---------------------------------------------- readComp(file=targetspath, format="vector", delim="-") ## ----param_structure, eval=TRUE------------------------------------------ parampath <- system.file("extdata", "tophat.param", package="systemPipeR") read.delim(parampath, comment.char = "#") ## ----param_import, eval=TRUE--------------------------------------------- args <- suppressWarnings(systemArgs(sysma=parampath, mytargets=targetspath)) args ## ----sysarg_access, eval=TRUE-------------------------------------------- names(args) ## ----sysarg_access2, eval=TRUE------------------------------------------- sysargs(args)[1] modules(args) cores(args) outpaths(args)[1] ## ----sysarg_json, eval=TRUE---------------------------------------------- systemArgs(sysma=parampath, mytargets=targetspath, type="json") ## ----load_package, eval=FALSE-------------------------------------------- ## library(systemPipeR) ## library(systemPipeRdata) ## genWorkenvir(workflow="rnaseq", mydirname=NULL) ## setwd("rnaseq") ## ----construct_sysargs, eval=FALSE--------------------------------------- ## args <- systemArgs(sysma="param/trim.param", mytargets="targets.txt") ## ----preprocessing, eval=FALSE------------------------------------------- ## preprocessReads(args=args, Fct="trimLRPatterns(Rpattern='GCCCGGGTAA', subject=fq)", ## batchsize=100000, overwrite=TRUE, compress=TRUE) ## writeTargetsout(x=args, file="targets_trim.txt") ## ----custom_preprocessing, eval=FALSE------------------------------------ ## args <- systemArgs(sysma="param/trimPE.param", mytargets="targetsPE.txt") ## filterFct <- function(fq, cutoff=20, Nexceptions=0) { ## qcount <- rowSums(as(quality(fq), "matrix") <= cutoff) ## fq[qcount <= Nexceptions] # Retains reads where Phred scores are >= cutoff with N exceptions ## } ## preprocessReads(args=args, Fct="filterFct(fq, cutoff=20, Nexceptions=0)", batchsize=100000) ## writeTargetsout(x=args, file="targets_PEtrim.txt") ## ----fastq_quality, eval=FALSE------------------------------------------- ## fqlist <- seeFastq(fastq=infile1(args), batchsize=10000, klength=8) ## pdf("./results/fastqReport.pdf", height=18, width=4*length(fqlist)) ## seeFastqPlot(fqlist) ## dev.off() ## ----fastq_quality_parallel_single, eval=FALSE--------------------------- ## args <- systemArgs(sysma="param/tophat.param", mytargets="targets.txt") ## f <- function(x) seeFastq(fastq=infile1(args)[x], batchsize=100000, klength=8) ## fqlist <- bplapply(seq(along=args), f, BPPARAM = MulticoreParam(workers=8)) ## seeFastqPlot(unlist(fqlist, recursive=FALSE)) ## ----fastq_quality_parallel_cluster, eval=FALSE-------------------------- ## library(BiocParallel); library(BatchJobs) ## f <- function(x) { ## library(systemPipeR) ## args <- systemArgs(sysma="param/tophat.param", mytargets="targets.txt") ## seeFastq(fastq=infile1(args)[x], batchsize=100000, klength=8) ## } ## funs <- makeClusterFunctionsTorque("torque.tmpl") ## param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs) ## register(param) ## fqlist <- bplapply(seq(along=args), f) ## seeFastqPlot(unlist(fqlist, recursive=FALSE)) ## ----bowtie_index, eval=FALSE-------------------------------------------- ## args <- systemArgs(sysma="param/tophat.param", mytargets="targets.txt") ## moduleload(modules(args)) # Skip if module system is not available ## system("bowtie2-build ./data/tair10.fasta ./data/tair10.fasta") ## ----run_bowtie_single, eval=FALSE--------------------------------------- ## bampaths <- runCommandline(args=args) ## ----run_bowtie_parallel, eval=FALSE------------------------------------- ## resources <- list(walltime="20:00:00", nodes=paste0("1:ppn=", cores(args)), memory="10gb") ## reg <- clusterRun(args, conffile=".BatchJobs.R", template="torque.tmpl", Njobs=18, runid="01", ## resourceList=resources) ## waitForJobs(reg) ## ----process_monitoring, eval=FALSE-------------------------------------- ## showStatus(reg) ## file.exists(outpaths(args)) ## sapply(1:length(args), function(x) loadResult(reg, x)) # Works after job completion ## ----align_stats1, eval=FALSE-------------------------------------------- ## read_statsDF <- alignStats(args) ## write.table(read_statsDF, "results/alignStats.xls", row.names=FALSE, quote=FALSE, sep="\t") ## ----align_stats2, eval=TRUE--------------------------------------------- read.table(system.file("extdata", "alignStats.xls", package="systemPipeR"), header=TRUE)[1:4,] ## ----align_stats_parallel, eval=FALSE------------------------------------ ## f <- function(x) alignStats(args[x]) ## read_statsList <- bplapply(seq(along=args), f, BPPARAM = MulticoreParam(workers=8)) ## read_statsDF <- do.call("rbind", read_statsList) ## ----align_stats_parallel_cluster, eval=FALSE---------------------------- ## library(BiocParallel); library(BatchJobs) ## f <- function(x) { ## library(systemPipeR) ## args <- systemArgs(sysma="tophat.param", mytargets="targets.txt") ## alignStats(args[x]) ## } ## funs <- makeClusterFunctionsTorque("torque.tmpl") ## param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs) ## register(param) ## read_statsList <- bplapply(seq(along=args), f) ## read_statsDF <- do.call("rbind", read_statsList) ## ----igv, eval=FALSE----------------------------------------------------- ## symLink2bam(sysargs=args, htmldir=c("~/.html/", "somedir/"), ## urlbase="http://myserver.edu/~username/", ## urlfile="IGVurl.txt") ## ----bowtie2, eval=FALSE------------------------------------------------- ## args <- systemArgs(sysma="bowtieSE.param", mytargets="targets.txt") ## moduleload(modules(args)) # Skip if module system is not available ## bampaths <- runCommandline(args=args) ## ----bowtie2_cluster, eval=FALSE----------------------------------------- ## resources <- list(walltime="20:00:00", nodes=paste0("1:ppn=", cores(args)), memory="10gb") ## reg <- clusterRun(args, conffile=".BatchJobs.R", template="torque.tmpl", Njobs=18, runid="01", ## resourceList=resources) ## waitForJobs(reg) ## ----bwamem_cluster, eval=FALSE------------------------------------------ ## args <- systemArgs(sysma="param/bwa.param", mytargets="targets.txt") ## moduleload(modules(args)) # Skip if module system is not available ## system("bwa index -a bwtsw ./data/tair10.fasta") # Indexes reference genome ## bampaths <- runCommandline(args=args[1:2]) ## ----rsubread, eval=FALSE------------------------------------------------ ## library(Rsubread) ## args <- systemArgs(sysma="param/rsubread.param", mytargets="targets.txt") ## buildindex(basename=reference(args), reference=reference(args)) # Build indexed reference genome ## align(index=reference(args), readfile1=infile1(args)[1:4], input_format="FASTQ", ## output_file=outfile1(args)[1:4], output_format="SAM", nthreads=8, indels=1, TH1=2) ## for(i in seq(along=outfile1(args))) asBam(file=outfile1(args)[i], destination=gsub(".sam", "", outfile1(args)[i]), overwrite=TRUE, indexDestination=TRUE) ## ----gsnap, eval=FALSE--------------------------------------------------- ## library(gmapR); library(BiocParallel); library(BatchJobs) ## args <- systemArgs(sysma="param/gsnap.param", mytargets="targetsPE.txt") ## gmapGenome <- GmapGenome(reference(args), directory="data", name="gmap_tair10chr/", create=TRUE) ## f <- function(x) { ## library(gmapR); library(systemPipeR) ## args <- systemArgs(sysma="gsnap.param", mytargets="targetsPE.txt") ## gmapGenome <- GmapGenome(reference(args), directory="data", name="gmap_tair10chr/", create=FALSE) ## p <- GsnapParam(genome=gmapGenome, unique_only=TRUE, molecule="DNA", max_mismatches=3) ## o <- gsnap(input_a=infile1(args)[x], input_b=infile2(args)[x], params=p, output=outfile1(args)[x]) ## } ## funs <- makeClusterFunctionsTorque("torque.tmpl") ## param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs) ## register(param) ## d <- bplapply(seq(along=args), f) ## ----create_txdb, eval=FALSE--------------------------------------------- ## library(GenomicFeatures) ## txdb <- makeTxDbFromGFF(file="data/tair10.gff", format="gff", dataSource="TAIR", organism="A. thaliana") ## saveDb(txdb, file="./data/tair10.sqlite") ## ----read_counting_multicore, eval=FALSE--------------------------------- ## library(BiocParallel) ## txdb <- loadDb("./data/tair10.sqlite") ## eByg <- exonsBy(txdb, by="gene") ## bfl <- BamFileList(outpaths(args), yieldSize=50000, index=character()) ## multicoreParam <- MulticoreParam(workers=4); register(multicoreParam); registered() ## counteByg <- bplapply(bfl, function(x) summarizeOverlaps(eByg, x, mode="Union", ignore.strand=TRUE, inter.feature=TRUE, singleEnd=TRUE)) # Note: for strand-specific RNA-Seq set 'ignore.strand=FALSE' and for PE data set 'singleEnd=FALSE' ## countDFeByg <- sapply(seq(along=counteByg), function(x) assays(counteByg[[x]])$counts) ## rownames(countDFeByg) <- names(rowRanges(counteByg[[1]])); colnames(countDFeByg) <- names(bfl) ## rpkmDFeByg <- apply(countDFeByg, 2, function(x) returnRPKM(counts=x, ranges=eByg)) ## write.table(countDFeByg, "results/countDFeByg.xls", col.names=NA, quote=FALSE, sep="\t") ## write.table(rpkmDFeByg, "results/rpkmDFeByg.xls", col.names=NA, quote=FALSE, sep="\t") ## ----read_counting_multinode, eval=FALSE--------------------------------- ## library(BiocParallel) ## f <- function(x) { ## library(systemPipeR); library(BiocParallel); library(GenomicFeatures) ## txdb <- loadDb("./data/tair10.sqlite") ## eByg <- exonsBy(txdb, by="gene") ## args <- systemArgs(sysma="tophat.param", mytargets="targets.txt") ## bfl <- BamFileList(outpaths(args), yieldSize=50000, index=character()) ## summarizeOverlaps(eByg, bfl[x], mode="Union", ignore.strand=TRUE, inter.feature=TRUE, singleEnd=TRUE) ## } ## funs <- makeClusterFunctionsTorque("torque.tmpl") ## param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs) ## register(param) ## counteByg <- bplapply(seq(along=args), f) ## countDFeByg <- sapply(seq(along=counteByg), function(x) assays(counteByg[[x]])$counts) ## rownames(countDFeByg) <- names(rowRanges(counteByg[[1]])); colnames(countDFeByg) <- names(outpaths(args)) ## ----read_counting_mirna, eval=FALSE------------------------------------- ## system("wget ftp://mirbase.org/pub/mirbase/19/genomes/My_species.gff3 -P ./data/") ## gff <- import.gff("./data/My_species.gff3") ## gff <- split(gff, elementMetadata(gff)$ID) ## bams <- names(bampaths); names(bams) <- targets$SampleName ## bfl <- BamFileList(bams, yieldSize=50000, index=character()) ## countDFmiR <- summarizeOverlaps(gff, bfl, mode="Union", ignore.strand=FALSE, inter.feature=FALSE) # Note: inter.feature=FALSE important since pre and mature miRNA ranges overlap ## rpkmDFmiR <- apply(countDFmiR, 2, function(x) returnRPKM(counts=x, gffsub=gff)) ## write.table(assays(countDFmiR)$counts, "results/countDFmiR.xls", col.names=NA, quote=FALSE, sep="\t") ## write.table(rpkmDFmiR, "results/rpkmDFmiR.xls", col.names=NA, quote=FALSE, sep="\t") ## ----sample_tree_rlog, eval=TRUE----------------------------------------- library(DESeq2, warn.conflicts=FALSE, quietly=TRUE); library(ape, warn.conflicts=FALSE) countDFpath <- system.file("extdata", "countDFeByg.xls", package="systemPipeR") countDF <- as.matrix(read.table(countDFpath)) colData <- data.frame(row.names=targetsin(args)$SampleName, condition=targetsin(args)$Factor) dds <- DESeqDataSetFromMatrix(countData = countDF, colData = colData, design = ~ condition) d <- cor(assay(rlog(dds)), method="spearman") hc <- hclust(dist(1-d)) plot.phylo(as.phylo(hc), type="p", edge.col=4, edge.width=3, show.node.label=TRUE, no.margin=TRUE) ## ----sample_tree_rpkm, eval=FALSE---------------------------------------- ## rpkmDFeBygpath <- system.file("extdata", "rpkmDFeByg.xls", package="systemPipeR") ## rpkmDFeByg <- read.table(rpkmDFeBygpath, check.names=FALSE) ## rpkmDFeByg <- rpkmDFeByg[rowMeans(rpkmDFeByg) > 50,] ## d <- cor(rpkmDFeByg, method="spearman") ## hc <- hclust(as.dist(1-d)) ## plot.phylo(as.phylo(hc), type="p", edge.col="blue", edge.width=2, show.node.label=TRUE, no.margin=TRUE) ## ----edger_wrapper, eval=TRUE-------------------------------------------- targets <- read.delim(targetspath, comment="#") cmp <- readComp(file=targetspath, format="matrix", delim="-") cmp[[1]] countDFeBygpath <- system.file("extdata", "countDFeByg.xls", package="systemPipeR") countDFeByg <- read.delim(countDFeBygpath, row.names=1) edgeDF <- run_edgeR(countDF=countDFeByg, targets=targets, cmp=cmp[[1]], independent=FALSE, mdsplot="") ## ----edger_deg_counts, eval=TRUE----------------------------------------- DEG_list <- filterDEGs(degDF=edgeDF, filter=c(Fold=2, FDR=10)) ## ----edger_deg_stats, eval=TRUE------------------------------------------ names(DEG_list) DEG_list$Summary[1:4,] ## ----deseq2_wrapper, eval=TRUE------------------------------------------- degseqDF <- run_DESeq2(countDF=countDFeByg, targets=targets, cmp=cmp[[1]], independent=FALSE) ## ----deseq2_deg_counts, eval=TRUE---------------------------------------- DEG_list2 <- filterDEGs(degDF=degseqDF, filter=c(Fold=2, FDR=10)) ## ----vennplot, eval=TRUE------------------------------------------------- vennsetup <- overLapper(DEG_list$Up[6:9], type="vennsets") vennsetdown <- overLapper(DEG_list$Down[6:9], type="vennsets") vennPlot(list(vennsetup, vennsetdown), mymain="", mysub="", colmode=2, ccol=c("blue", "red")) ## ----get_go_biomart, eval=FALSE------------------------------------------ ## library("biomaRt") ## listMarts() # To choose BioMart database ## m <- useMart("ENSEMBL_MART_PLANT"); listDatasets(m) ## m <- useMart("ENSEMBL_MART_PLANT", dataset="athaliana_eg_gene") ## listAttributes(m) # Choose data types you want to download ## go <- getBM(attributes=c("go_accession", "tair_locus", "go_namespace_1003"), mart=m) ## go <- go[go[,3]!="",]; go[,3] <- as.character(go[,3]) ## dir.create("./data/GO") ## write.table(go, "data/GO/GOannotationsBiomart_mod.txt", quote=FALSE, row.names=FALSE, col.names=FALSE, sep="\t") ## catdb <- makeCATdb(myfile="data/GO/GOannotationsBiomart_mod.txt", lib=NULL, org="", colno=c(1,2,3), idconv=NULL) ## save(catdb, file="data/GO/catdb.RData") ## ----go_enrichment, eval=FALSE------------------------------------------- ## load("data/GO/catdb.RData") ## DEG_list <- filterDEGs(degDF=edgeDF, filter=c(Fold=2, FDR=50), plot=FALSE) ## up_down <- DEG_list$UporDown; names(up_down) <- paste(names(up_down), "_up_down", sep="") ## up <- DEG_list$Up; names(up) <- paste(names(up), "_up", sep="") ## down <- DEG_list$Down; names(down) <- paste(names(down), "_down", sep="") ## DEGlist <- c(up_down, up, down) ## DEGlist <- DEGlist[sapply(DEGlist, length) > 0] ## BatchResult <- GOCluster_Report(catdb=catdb, setlist=DEGlist, method="all", id_type="gene", CLSZ=2, cutoff=0.9, gocats=c("MF", "BP", "CC"), recordSpecGO=NULL) ## library("biomaRt"); m <- useMart("ENSEMBL_MART_PLANT", dataset="athaliana_eg_gene") ## goslimvec <- as.character(getBM(attributes=c("goslim_goa_accession"), mart=m)[,1]) ## BatchResultslim <- GOCluster_Report(catdb=catdb, setlist=DEGlist, method="slim", id_type="gene", myslimv=goslimvec, CLSZ=10, cutoff=0.01, gocats=c("MF", "BP", "CC"), recordSpecGO=NULL) ## ----plot_go_enrichment, eval=FALSE-------------------------------------- ## gos <- BatchResultslim[grep("M6-V6_up_down", BatchResultslim$CLID), ] ## gos <- BatchResultslim ## pdf("GOslimbarplotMF.pdf", height=8, width=10); goBarplot(gos, gocat="MF"); dev.off() ## goBarplot(gos, gocat="BP") ## goBarplot(gos, gocat="CC") ## ----hierarchical_clustering, eval=FALSE--------------------------------- ## library(pheatmap) ## geneids <- unique(as.character(unlist(DEG_list[[1]]))) ## y <- assay(rlog(dds))[geneids, ] ## pdf("heatmap1.pdf") ## pheatmap(y, scale="row", clustering_distance_rows="correlation", clustering_distance_cols="correlation") ## dev.off() ## ----genRna_workflow_single, eval=FALSE---------------------------------- ## library(systemPipeRdata) ## genWorkenvir(workflow="rnaseq") ## setwd("rnaseq") ## ----genChip_workflow_single, eval=FALSE--------------------------------- ## library(systemPipeRdata) ## genWorkenvir(workflow="chipseq") ## setwd("chipseq") ## ----genVar_workflow_single, eval=FALSE---------------------------------- ## library(systemPipeRdata) ## genWorkenvir(workflow="varseq") ## setwd("varseq") ## ----genRibo_workflow_single, eval=FALSE--------------------------------- ## library(systemPipeRdata) ## genWorkenvir(workflow="riboseq") ## setwd("riboseq") ## ----sessionInfo--------------------------------------------------------- sessionInfo()