## ----options, results='hide', echo=FALSE-----------------------------------
#options(width=65)
options('useFancyQuotes' = FALSE, continue=" ", digits=3)

## ----cite, eval=TRUE-------------------------------------------------------
citation("QuasR")

## ----install, eval=FALSE---------------------------------------------------
#  if (!require("BiocManager"))
#      install.packages("BiocManager")
#  BiocManager::install("QuasR")

## ----loadLibraries, eval=TRUE----------------------------------------------
library(QuasR)
library(BSgenome)
library(Rsamtools)
library(rtracklayer)
library(GenomicFeatures)
library(Gviz)

## ----help1, eval=FALSE-----------------------------------------------------
#  help.start()

## ----loadQuasRLibrary, eval=FALSE------------------------------------------
#  library(QuasR)

## ----help2, eval=FALSE-----------------------------------------------------
#  ?preprocessReads

## ----help3, eval=FALSE-----------------------------------------------------
#  help("preprocessReads")

## ----assign, eval=FALSE----------------------------------------------------
#  x <- 2

## ----ls, eval=FALSE--------------------------------------------------------
#  ls()

## ----printObject, eval=FALSE-----------------------------------------------
#  x

## ----SampleSession1, eval=TRUE---------------------------------------------
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)

## ----SampleSession2, eval=TRUE---------------------------------------------
sampleFile <- "extdata/samples_chip_single.txt"
genomeFile <- "extdata/hg19sub.fa"

proj <- qAlign(sampleFile, genomeFile)
proj

## ----SampleSession3, eval=TRUE---------------------------------------------
qQCReport(proj, "extdata/qc_report.pdf")

## ----SampleSession4, eval=TRUE---------------------------------------------
library(rtracklayer)
library(GenomicFeatures)
annotFile <- "extdata/hg19sub_annotation.gtf"
txStart <- import.gff(annotFile, format="gtf", feature.type="start_codon")
promReg <- promoters(txStart, upstream=500, downstream=500)
names(promReg) <- mcols(promReg)$transcript_name

promCounts <- qCount(proj, query=promReg)
promCounts

## ----sampleFileSingle, echo=FALSE, results="asis"--------------------------
cat(paste(readLines(system.file(package="QuasR", "extdata", "samples_chip_single.txt")),
          collapse="\n"))

## ----sampleFilePaired, echo=FALSE, results="asis"--------------------------
cat(paste(readLines(system.file(package="QuasR", "extdata", "samples_rna_paired.txt")),
      collapse="\n"))

## ----sampleFile, eval=FALSE------------------------------------------------
#  sampleFile1 <- system.file(package="QuasR", "extdata",
#                             "samples_chip_single.txt")
#  sampleFile2 <- system.file(package="QuasR", "extdata",
#                             "samples_rna_paired.txt")

## ----<sampleFileSeqToBam, eval=FALSE---------------------------------------
#  sampleFile1 <- "samples_fastq.txt"
#  sampleFile2 <- "samples_bam.txt"
#  
#  proj1 <- qAlign(sampleFile1, genomeFile)
#  
#  write.table(alignments(proj1)$genome, sampleFile2, sep="\t", row.names=FALSE)
#  
#  proj2 <- qAlign(sampleFile2, genomeFile)

## ----auxFile, echo=FALSE, results="asis"-----------------------------------
cat(paste(readLines(system.file(package="QuasR", "extdata", "auxiliaries.txt")),
          collapse="\n"))

## ----auxiliaryFile, eval=TRUE----------------------------------------------
auxFile <- system.file(package="QuasR", "extdata", "auxiliaries.txt")

## ----selectGenomeBSgenome, eval=TRUE---------------------------------------
available.genomes()
genomeName <- "BSgenome.Hsapiens.UCSC.hg19"

## ----selectGenomeFile, eval=FALSE------------------------------------------
#  genomeFile <- system.file(package="QuasR", "extdata", "hg19sub.fa")

## ----preprocessReadsSingle,eval=TRUE---------------------------------------
td <- tempdir()
infiles <- system.file(package="QuasR", "extdata",
                       c("rna_1_1.fq.bz2","rna_2_1.fq.bz2"))
outfiles <- file.path(td, basename(infiles))
res <- preprocessReads(filename = infiles,
                       outputFilename = outfiles,
                       truncateEndBases = 3,
                       Lpattern = "AAAAAAAAAA",
                       minLength = 14, 
                       nBases = 2)
res
unlink(outfiles)

## ----preprocessReadsPaired,eval=TRUE---------------------------------------
td <- tempdir()
infiles1 <- system.file(package="QuasR", "extdata", "rna_1_1.fq.bz2")
infiles2 <- system.file(package="QuasR", "extdata", "rna_1_2.fq.bz2")
outfiles1 <- file.path(td, basename(infiles1))
outfiles2 <- file.path(td, basename(infiles2))
res <- preprocessReads(filename=infiles1,
                       filenameMate=infiles2,
                       outputFilename=outfiles1,
                       outputFilenameMate=outfiles2,
                       nBases=0)
res

## ----ChIP_copyExtdata, eval=TRUE-------------------------------------------
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)

## ----ChIP_qAlign, eval=TRUE------------------------------------------------
sampleFile <- "extdata/samples_chip_single.txt"
auxFile <- "extdata/auxiliaries.txt"
genomeFile <- "extdata/hg19sub.fa"

proj1 <- qAlign(sampleFile, genome=genomeFile, auxiliaryFile=auxFile)
proj1

## ----ChIP_bamfiles1, eval=TRUE---------------------------------------------
list.files("extdata", pattern=".bam$")

## ----ChIP_bamfiles2, eval=TRUE---------------------------------------------
list.files("extdata", pattern="^chip_1_1_")[1:3]

## ----ChIP_qcplot1, eval=TRUE, echo=FALSE-----------------------------------
qcdat1 <- qQCReport(proj1, pdfFilename="extdata/qc_report.pdf")

## ----ChIP_qcplots2, eval=TRUE----------------------------------------------
qQCReport(proj1, pdfFilename="extdata/qc_report.pdf")

## ----ChIP_alignmentStats, eval=TRUE----------------------------------------
alignmentStats(proj1)

## ----ChIP_qExportWig, eval=TRUE--------------------------------------------
qExportWig(proj1, binsize=100L, scaling=TRUE, collapseBySample=TRUE)

## ----ChIP_GenomicFeatures, eval=TRUE---------------------------------------
library(GenomicFeatures)
annotFile <- "extdata/hg19sub_annotation.gtf"
chrLen <- scanFaIndex(genomeFile)
chrominfo <- data.frame(chrom=as.character(seqnames(chrLen)),
                        length=width(chrLen),
                        is_circular=rep(FALSE, length(chrLen)))
txdb <- makeTxDbFromGFF(file=annotFile, format="gtf",
                        chrominfo=chrominfo,
                        dataSource="Ensembl",
                        organism="Homo sapiens")
promReg <- promoters(txdb, upstream=1000, downstream=500,
                     columns=c("gene_id","tx_id"))
gnId <- sapply(mcols(promReg)$gene_id, paste, collapse=",")
promRegSel <- promReg[ match(unique(gnId), gnId) ]
names(promRegSel) <- unique(gnId)
head(promRegSel)

## ----ChIP_qCount, eval=TRUE------------------------------------------------
cnt <- qCount(proj1, promRegSel)
cnt

## ----ChIP_visualize, eval=TRUE, fig.width=8, fig.height=4.5----------------
gr1 <- import("Sample1.wig.gz")
gr2 <- import("Sample2.wig.gz")

library(Gviz)
axisTrack <- GenomeAxisTrack()
dTrack1 <- DataTrack(range=gr1, name="Sample 1", type="h")
dTrack2 <- DataTrack(range=gr2, name="Sample 2", type="h")
txTrack <- GeneRegionTrack(txdb, name="Transcripts", showId=TRUE)
plotTracks(list(axisTrack, dTrack1, dTrack2, txTrack),
           chromosome="chr3", extend.left=1000)

## ----ChIP_rtracklayer, eval=TRUE-------------------------------------------
library(rtracklayer)
annotationFile <- "extdata/hg19sub_annotation.gtf"
tssRegions <- import.gff(annotationFile, format="gtf",
                         feature.type="start_codon",
                         colnames="gene_name")
tssRegions <- tssRegions[!duplicated(tssRegions)]
names(tssRegions) <- rep("TSS", length(tssRegions))
head(tssRegions)

## ----ChIP_qProfile, eval=TRUE----------------------------------------------
prS <- qProfile(proj1, tssRegions, upstream=3000, downstream=3000, 
                orientation="same")
prO <- qProfile(proj1, tssRegions, upstream=3000, downstream=3000, 
                orientation="opposite")
lapply(prS, "[", , 1:10)

## ----ChIP_visualizeProfile, eval=TRUE, fig.width=8, fig.height=4.5---------
prCombS <- do.call("+", prS[-1]) /prS[[1]]
prCombO <- do.call("+", prO[-1]) /prO[[1]]

plot(as.numeric(colnames(prCombS)), filter(prCombS[1,], rep(1/100,100)), 
     type='l', xlab="Position relative to TSS", ylab="Mean no. of alignments")
lines(as.numeric(colnames(prCombO)), filter(prCombO[1,], rep(1/100,100)), 
      type='l', col="red")
legend(title="strand", legend=c("same as query","opposite of query"), 
       x="topleft", col=c("black","red"), lwd=1.5, bty="n", title.adj=0.1)

## ----ChIP_BSgenomeProject, eval=FALSE--------------------------------------
#  file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)
#  
#  sampleFile <- "extdata/samples_chip_single.txt"
#  auxFile <- "extdata/auxiliaries.txt"
#  
#  available.genomes() # list available genomes
#  genomeName <- "BSgenome.Hsapiens.UCSC.hg19"
#  
#  proj1 <- qAlign(sampleFile, genome=genomeName, auxiliaryFile=auxFile)
#  proj1

## ----RNA_qAlign, eval=TRUE-------------------------------------------------
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)

sampleFile <- "extdata/samples_rna_paired.txt"
genomeFile <- "extdata/hg19sub.fa"

proj2 <- qAlign(sampleFile, genome=genomeFile, splicedAlignment=TRUE)
proj2

## ----RNA_alignmentStats, eval=TRUE-----------------------------------------
proj2unspl <- qAlign(sampleFile, genome=genomeFile,
                     splicedAlignment=FALSE)

alignmentStats(proj2)
alignmentStats(proj2unspl)

## ----RNA_qCount, eval=TRUE-------------------------------------------------
geneLevels <- qCount(proj2, txdb, reportLevel="gene")
exonLevels <- qCount(proj2, txdb, reportLevel="exon")

head(geneLevels)
head(exonLevels)

## ----RNA_RPMK, eval=TRUE---------------------------------------------------
geneRPKM <- t(t(geneLevels[,-1] /geneLevels[,1] *1000)
              /colSums(geneLevels[,-1]) *1e6)
geneRPKM

## ----RNA_junction, eval=TRUE-----------------------------------------------
exonJunctions <- qCount(proj2, NULL, reportLevel="junction")
exonJunctions

## ----RNA_junction2, eval=TRUE----------------------------------------------
knownIntrons <- unlist(intronsByTranscript(txdb))
isKnown <- overlapsAny(exonJunctions, knownIntrons, type="equal")
table(isKnown)

tapply(rowSums(as.matrix(mcols(exonJunctions))),
       isKnown, summary)

## ----RNA_includeSpliced, eval=TRUE-----------------------------------------
exonBodyLevels <- qCount(proj2, txdb, reportLevel="exon", includeSpliced=FALSE)
summary(exonLevels - exonBodyLevels)

## ----RNA_qcplot1, eval=TRUE, echo=FALSE------------------------------------
qcdat2 <- qQCReport(proj2unspl, pdfFilename="qc_report.pdf")

## ----miRNA_extdata, eval=TRUE----------------------------------------------
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)

## ----miRNA_preprocessReads, eval=TRUE--------------------------------------
# prepare sample file with processed reads filenames
sampleFile <- file.path("extdata", "samples_mirna.txt")
sampleFile
sampleFile2 <- sub(".txt", "_processed.txt", sampleFile)
sampleFile2

tab <- read.delim(sampleFile, header=TRUE, as.is=TRUE)
tab

tab2 <- tab
tab2$FileName <- sub(".fa", "_processed.fa", tab$FileName)
write.table(tab2, sampleFile2, sep="\t", quote=FALSE, row.names=FALSE)
tab2

# remove adapters
oldwd <- setwd(dirname(sampleFile))
res <- preprocessReads(tab$FileName,
                       tab2$FileName,
                       Rpattern="TGGAATTCTCGGGTGCCAAGG")
res
setwd(oldwd)

## ----miRNA_lengthes, eval=TRUE, fig.width=6, fig.height=4.5----------------
# get read lengths
library(Biostrings)
oldwd <- setwd(dirname(sampleFile))
lens <- fasta.seqlengths(tab$FileName, nrec=1e5)
lens2 <- fasta.seqlengths(tab2$FileName, nrec=1e5)
setwd(oldwd)
# plot length distribution
lensTab <- rbind(raw=tabulate(lens,50),
                 processed=tabulate(lens2,50))
colnames(lensTab) <- 1:50
barplot(lensTab/rowSums(lensTab)*100,
        xlab="Read length (nt)", ylab="Percent of reads")
legend(x="topleft", bty="n", fill=gray.colors(2), legend=rownames(lensTab))

## ----miRNA_qAlign, eval=TRUE-----------------------------------------------
proj3 <- qAlign(sampleFile2, genomeFile, maxHits=50)
alignmentStats(proj3)

## ----miRNA_prepareQuery, eval=TRUE-----------------------------------------
mirs <- import("extdata/mirbaseXX_qsr.gff3")
names(mirs) <- mirs$Name
preMirs <- mirs[ mirs$type=="miRNA_primary_transcript" ]
matureMirs <- mirs[ mirs$type=="miRNA" ]

## ----miRNA_coverage, eval=TRUE, fig.width=6, fig.height=4.5----------------
library(Rsamtools)
alns <- scanBam(alignments(proj3)$genome$FileName,
                param=ScanBamParam(what=scanBamWhat(), which=preMirs[1]))[[1]]
alnsIR <- IRanges(start=alns$pos - start(preMirs), width=alns$qwidth)
mp <- barplot(as.vector(coverage(alnsIR)), names.arg=1:max(end(alnsIR)),
              xlab="Relative position in pre-miRNA",
              ylab="Alignment coverage")
rect(xleft=mp[start(matureMirs)-start(preMirs)+1,1], ybottom=-par('cxy')[2],
     xright=mp[end(matureMirs)-start(preMirs)+1,1], ytop=0,
     col="#CCAA0088", border=NA, xpd=NA)

## ----miRNA_extendQuery, eval=TRUE------------------------------------------
matureMirsExtended <- resize(matureMirs, width=1L, fix="start") + 3L

## ----miRNA_quantify, eval=TRUE---------------------------------------------
# quantify mature miRNAs
cnt <- qCount(proj3, matureMirsExtended, orientation="same")
cnt

# quantify pre-miRNAs
cnt <- qCount(proj3, preMirs, orientation="same")
cnt

## ----Bis_qAlign, eval=TRUE-------------------------------------------------
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)

sampleFile <- "extdata/samples_bis_single.txt"
genomeFile <- "extdata/hg19sub.fa"

proj4 <- qAlign(sampleFile, genomeFile, bisulfite="dir")
proj4

## ----Bis_qMeth1, eval=TRUE-------------------------------------------------
meth <- qMeth(proj4, mode="CpGcomb", collapseBySample=TRUE)
meth

## ----Bis_qMeth2, eval=TRUE-------------------------------------------------
chrs <- readDNAStringSet(genomeFile)
sum(vcountPattern("CG",chrs))
length(qMeth(proj4))
length(qMeth(proj4, keepZero=FALSE))

## ----Bis_visualize, eval=TRUE, fig.height=4.5, fig.width=8-----------------
percMeth <- mcols(meth)[,2] *100 /mcols(meth)[,1]
summary(percMeth)

axisTrack <- GenomeAxisTrack()
dTrack1 <- DataTrack(range=gr1, name="H3K4me3", type="h")
dTrack2 <- DataTrack(range=meth, data=percMeth,
                     name="Methylation", type="p")
txTrack <- GeneRegionTrack(txdb, name="Transcripts", showId=TRUE)
plotTracks(list(axisTrack, dTrack1, dTrack2, txTrack),
           chromosome="chr3", extend.left=1000)

## ----Bis_query, eval=TRUE--------------------------------------------------
qMeth(proj4, query=GRanges("chr1",IRanges(start=31633,width=2)),
      collapseBySample=TRUE)
qMeth(proj4, query=promRegSel, collapseByQueryRegion=TRUE,
      collapseBySample=TRUE)

## ----snpFile, echo=FALSE, results="asis"-----------------------------------
cat(paste(c(readLines(system.file(package="QuasR", "extdata", "hg19sub_snp.txt"))[1:4], "..."),
          collapse="\n"))

## ----Alelle_Extdata, eval=TRUE---------------------------------------------
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)

## ----Allele_qAlign, eval=TRUE----------------------------------------------
sampleFile <- "extdata/samples_chip_single.txt"
genomeFile <- "extdata/hg19sub.fa"
snpFile <- "extdata/hg19sub_snp.txt"
proj1SNP <- qAlign(sampleFile, genome=genomeFile, snpFile=snpFile)
proj1SNP

## ----Allele_qCount, eval=TRUE----------------------------------------------
head(qCount(proj1, promRegSel))
head(qCount(proj1SNP, promRegSel))

## ----Allele_Bis, eval=TRUE-------------------------------------------------
sampleFile <- "extdata/samples_bis_single.txt"
genomeFile <- "extdata/hg19sub.fa"
proj4SNP <- qAlign(sampleFile, genomeFile,
                   snpFile=snpFile, bisulfite="dir")
head(qMeth(proj4SNP, mode="CpGcomb", collapseBySample=TRUE))

## ----qcplotsFig1, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotQualByCycle(qcdat1$raw$qa, lmat=rbind(1:2))

## ----qcplotsFig2, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotNuclByCycle(qcdat1$raw$qa, lmat=rbind(1:2))

## ----qcplotsFig3, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotDuplicated(qcdat1$raw$qa, lmat=rbind(1:2))

## ----qcplotsFig4, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotMappings(qcdat1$raw$mapdata, a4layout=FALSE)

## ----qcplotsFig5, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotUniqueness(qcdat1$raw$unique, a4layout=FALSE)

## ----qcplotsFig6, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotErrorsByCycle(qcdat1$raw$mm, lmat=rbind(1:2))

## ----qcplotsFig7, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotMismatchTypes(qcdat1$raw$mm, lmat=rbind(1:2))

## ----qcplotsFig8, eval=TRUE, echo=FALSE, fig.height=4, fig.width=8---------
QuasR:::plotFragmentDistribution(qcdat2$raw$frag, lmat=rbind(1:2))

## ----alignmentStats, eval=TRUE---------------------------------------------
# using bam files
alignmentStats(alignments(proj1)$genome$FileName)
alignmentStats(unlist(alignments(proj1)$aux))

# using a qProject object
alignmentStats(proj1)

## ----sessionInfo, results='asis', echo=FALSE-------------------------------
toLatex(sessionInfo())

## ----cleanUp, eval=TRUE, echo=FALSE----------------------------------------
unlink("extdata", recursive=TRUE, force=TRUE)