Objectives
In this exercise, you will use a recently developed R package called RIPSeeker to identify binding regions in a small CLIP-seq dataset.
Primary Data
Ameyar-zazoua M, Rachez C, Souidi M, et al. Argonaute proteins couple chromatin silencing to alternative splicing. Nat Struct Mol Biol. 2012;19(10):998-1004.
RIPSeeker
RIPSeeker is a Bioconductor R package, and we are going to run the job using the "Rscript" utility using the script and commands file that exist in the directory you just staged. Check out the "ripseeker_script.R" file.
source("http://bioconductor.org/biocLite.R")
biocLite("RIPSeeker")
library(RIPSeeker)
bamFiles = list.files(".", "\\.bam$", recursive=TRUE, full.names=TRUE)
ripGal=combineAlignGals(bamFiles[2], genomeBuild="hg19")
ctlGal=combineAlignGals(bamFiles[1], genomeBuild="hg19")
cNAME="input"
genomeBuild = "hg19"
outDir <- file.path(getwd())
biomart="ensembl"
biomaRt_dataset="hsapiens_gene_ensembl"
goAnno="org.Hs.eg.db"
reverseComplement = FALSE
uniqueHit = FALSE
assignMultihits = TRUE
rerunWithDisambiguatedMultihits = TRUE
seekOut.AGO2 <- ripSeek(bamPath = bamFiles, cNAME = cNAME, reverseComplement = FALSE, genomeBuild = "hg19", uniqueHit = TRUE, assignMultihits = TRUE, rerunWithDisambiguatedMultihits = FALSE, binSize=binSize, biomart=biomart, biomaRt_dataset = biomaRt_dataset, goAnno = goAnno, outDir=outDir)
Now, before running anything, let's go through this section by section. First, we need to get the RIPSeeker packages from Bioconductor and load them into R.
> source("http://bioconductor.org/biocLite.R")
> biocLite("RIPSeeker")
> library(RIPSeeker)
Then, we need to read in the BAM files we're going to use with their paths and use the RIPSeeker (or rather GenomicRanges) function "combineAlignGals" to generate a "gapped alignment" object in R.
> bamFiles = list.files(".", "\\.bam$", recursive=TRUE, full.names=TRUE)
> ripGal=combineAlignGals(bamFiles[2], genomeBuild="hg19")
Processing ./ip.sort.bam ... All hits are returned with flags.
1 BAM files are combined
> ctlGal=combineAlignGals(bamFiles[1], genomeBuild="hg19")
Processing ./input.sort.bam ... All hits are returned with flags.
1 BAM files are combined
Now, we're ready to execute the primary RIPSeeker function, appropriately called ripSeek(). Since it takes so many options, we're going to declare a bunch of variables before executing the command, and then have the command refer back to those already-declared variables. This is what it would look like:
> cNAME="input" #Which files are input > genomeBuild = "hg19" #Genome to use > outDir <- file.path(getwd()) #Output file prefix (here, the current directory) > biomart="ensembl" #Source of genome annotations > biomaRt_dataset="hsapiens_gene_ensembl" #Specific dataset from biomaRt source > goAnno="org.Hs.eg.db" #Annotation database > reverseComplement = FALSE #Do not reverse complement BAM entries > uniqueHit = FALSE #If TRUE, use only unique hits to train initial model > assignMultihits = TRUE #If TRUE and uniqueHit is TRUE, assign multi-hits to regions based on initial model > rerunWithDisambiguatedMultihits = TRUE #If TRUE and prior two options are TRUE, re-train model after multi-hit assignment > seekOut.AGO2 <- ripSeek(bamPath = bamFiles, cNAME = cNAME, reverseComplement = FALSE, genomeBuild = "hg19", uniqueHit = TRUE, assignMultihits = TRUE, rerunWithDisambiguatedMultihits = FALSE, binSize=200, biomart=biomart, biomaRt_dataset = biomaRt_dataset, goAnno = goAnno, outDir=outDir)
If we execute the ripSeek() command, we get an output that looks something like this:
source("http://bioconductor.org/biocLite.R")
biocLite("RIPSeeker")
library(RIPSeeker)
bamFiles = list.files(".", "\\.bam$", recursive=TRUE, full.names=TRUE)
ripGal=combineAlignGals(bamFiles[2], genomeBuild="hg19")
ctlGal=combineAlignGals(bamFiles[1], genomeBuild="hg19")
cNAME="input"
genomeBuild = "hg19"
binSize=200
outDir <- file.path(getwd())
biomart="ensembl"
biomaRt_dataset="hsapiens_gene_ensembl"
goAnno="org.Hs.eg.db"
reverseComplement = FALSE
uniqueHit = FALSE
assignMultihits = TRUE
rerunWithDisambiguatedMultihits = TRUE
seekOut.AGO2 <- ripSeek(bamPath = bamFiles, cNAME = cNAME, reverseComplement = FALSE, genomeBuild = "hg19", uniqueHit = TRUE, assignMultihits = TRUE, rerunWithDisambiguatedMultihits = FALSE, binSize=binSize, biomart=biomart, biomaRt_dataset = biomaRt_dataset, goAnno = goAnno, outDir=outDir)