inst/doc/InPAS.R

In InPAS: InPAS: a bioconductor package for the identification of novel alternative PolyAdenylation Sites (PAS) using RNA-seq data

## ----preloadLibrary, echo=FALSE, results="hide", warning=FALSE----------------
suppressPackageStartupMessages({
    library(InPAS)
    library(BSgenome.Mmusculus.UCSC.mm10)
    library(TxDb.Mmusculus.UCSC.mm10.knownGene)
    library(org.Hs.eg.db)
    library(cleanUpdTSeq)
})

## ----loadLibrary--------------------------------------------------------------
library(InPAS)
library(BSgenome.Mmusculus.UCSC.mm10)
library(TxDb.Mmusculus.UCSC.mm10.knownGene)
path <- file.path(find.package("InPAS"), "extdata")

## ----prepareAnno--------------------------------------------------------------
library(org.Hs.eg.db)
samplefile <- system.file("extdata", "hg19_knownGene_sample.sqlite",
            package="GenomicFeatures")
txdb <- loadDb(samplefile)
utr3.sample.anno <- utr3Annotation(txdb=txdb, 
                   orgDbSYMBOL="org.Hs.egSYMBOL")
utr3.sample.anno

## ----loadAnno-----------------------------------------------------------------
##step1 annotation
# load from dataset
data(utr3.mm10)

## ----step2HugeDataF-----------------------------------------------------------
load(file.path(path, "polyA.rds"))
library(cleanUpdTSeq)
data(classifier)
bedgraphs <- c(file.path(path, "Baf3.extract.bedgraph"), 
               file.path(path, "UM15.extract.bedgraph"))
hugeData <- FALSE
##step1 Calculate coverage
coverage <- coverageFromBedGraph(bedgraphs, 
                                 tags=c("Baf3", "UM15"),
                                 genome=BSgenome.Mmusculus.UCSC.mm10,
                                 hugeData=hugeData)

## we hope the coverage rate of should be greater than 0.75 in the expressed gene.
## which is used because the demo data is a subset of genome.
coverageRate(coverage=coverage,
             txdb=TxDb.Mmusculus.UCSC.mm10.knownGene,
             genome=BSgenome.Mmusculus.UCSC.mm10,
             which = GRanges("chr6", ranges=IRanges(98013000, 140678000)))

##step2 Predict cleavage sites
CPs <- CPsites(coverage=coverage, 
               genome=BSgenome.Mmusculus.UCSC.mm10,
               utr3=utr3.mm10, 
               search_point_START=200, 
               cutEnd=.2, 
               long_coverage_threshold=3,
               background="10K",
               txdb=TxDb.Mmusculus.UCSC.mm10.knownGene,
               PolyA_PWM=pwm, 
               classifier=classifier,
               shift_range=50,
               step=10)
head(CPs)
##step3 Estimate 3UTR usage
res <- testUsage(CPsites=CPs, 
                 coverage=coverage, 
                 genome=BSgenome.Mmusculus.UCSC.mm10,
                 utr3=utr3.mm10, 
                 method="fisher.exact",
                 gp1="Baf3", gp2="UM15")

##step4 view the results
as(res, "GRanges") 

filterRes(res, gp1="Baf3", gp2="UM15",
          background_coverage_threshold=3,
          adj.P.Val_cutoff=0.05, 
          dPDUI_cutoff=0.3, 
          PDUI_logFC_cutoff=0.59)

## ----OneStep------------------------------------------------------------------
if(interactive()){
    res <- inPAS(bedgraphs=bedgraphs, tags=c("Baf3", "UM15"), 
              genome=BSgenome.Mmusculus.UCSC.mm10, 
              utr3=utr3.mm10, gp1="Baf3", gp2="UM15",
              txdb=TxDb.Mmusculus.UCSC.mm10.knownGene,
              search_point_START=200,
              short_coverage_threshold=15,
              long_coverage_threshold=3, 
              cutStart=0, cutEnd=.2, 
              hugeData=FALSE,
              shift_range=50,
              PolyA_PWM=pwm, classifier=classifier,
              method="fisher.exact",
              adj.P.Val_cutoff=0.05,
              dPDUI_cutoff=0.3, 
              PDUI_logFC_cutoff=0.59)
}

## ----SingleGroupData----------------------------------------------------------
inPAS(bedgraphs=bedgraphs[1], tags=c("Baf3"), 
      genome=BSgenome.Mmusculus.UCSC.mm10, 
      utr3=utr3.mm10, gp1="Baf3", gp2=NULL,
      txdb=TxDb.Mmusculus.UCSC.mm10.knownGene,
      search_point_START=200,
      short_coverage_threshold=15,
      long_coverage_threshold=3, 
      cutStart=0, cutEnd=.2, 
      hugeData=FALSE, 
      PolyA_PWM=pwm, classifier=classifier,
      method="singleSample",
      adj.P.Val_cutoff=0.05,
      step=10)

## ----sessionInfo, results='asis'----------------------------------------------
sessionInfo()