filterOffTarget: filter off targets and generate reports.

In CRISPRseek: Design of target-specific guide RNAs in CRISPR-Cas9, genome-editing systems

Description

filter off targets that meet the criteria set by users such as minimum score, topN. In addition, off target was annotated with flank sequence, gRNA cleavage efficiency and whether it is inside an exon or not if fetchSequence is set to TRUE and annotateExon is set to TRUE

Usage

filterOffTarget(scores, min.score = 0.01, topN = 200, 
    topN.OfftargetTotalScore = 20,
    annotateExon = TRUE, txdb, orgAnn,ignore.strand = TRUE,
    outputDir, oneFilePergRNA = FALSE, 
    fetchSequence = TRUE, upstream = 200, downstream = 200, BSgenomeName,
    baseBeforegRNA = 4, baseAfterPAM = 3,
    featureWeightMatrixFile = system.file("extdata", "DoenchNBT2014.csv", 
	package = "CRISPRseek"),
    rule.set = c("Root_RuleSet1_2014", "Root_RuleSet2_2016", "CRISPRscan"),
    calculategRNAefficacyForOfftargets = TRUE)

Arguments

scores	a data frame output from getOfftargetScore. It contains strand (strand of the off target, + for plus and - for minus strand), chrom (chromosome of the off target), chromStart (start position of the off target), chromEnd (end position of the off target),name (gRNA name),gRNAPlusPAM (gRNA sequence with PAM sequence concatenated), OffTargetSequence (the genomic sequence of the off target), n.mismatch (number of mismatches between the off target and the gRNA), forViewInUCSC (string for viewing in UCSC genome browser, e.g., chr14:31665685-31665707), score (score of the off target), mismatch.distance2PAM (a comma separated distances of all mismatches to PAM, e.g., 14,11 means one mismatch is 14 bp away from PAM and the other mismatch is 11 bp away from PAM), alignment (alignment between gRNA and off target, e.g., ......G..C.......... means that this off target aligns with gRNA except that G and C are mismatches),NGG (this off target contains canonical PAM or not, 1 for yes and 0 for no) mean.neighbor.distance.mismatch (mean distance between neighboring mismatches)
min.score	minimum score of an off target to included in the final output, default 0.5
topN	top N off targets to be included in the final output, default 100
topN.OfftargetTotalScore	top N off target used to calculate the total off target score, default 10
annotateExon	Choose whether or not to indicate whether the off target is inside an exon or not, default TRUE
txdb	TxDb object, for creating and using TxDb object, please refer to GenomicFeatures package. For a list of existing TxDb object, please search for annotation package starting with Txdb at http://www.bioconductor.org/packages/release/BiocViews.html#___AnnotationData, such as TxDb.Rnorvegicus.UCSC.rn5.refGene for rat, TxDb.Mmusculus.UCSC.mm10.knownGene for mouse, TxDb.Hsapiens.UCSC.hg19.knownGene for human, TxDb.Dmelanogaster.UCSC.dm3.ensGene for Drosophila and TxDb.Celegans.UCSC.ce6.ensGene for C.elegans
orgAnn	organism annotation mapping such as org.Hs.egSYMBOL in org.Hs.eg.db package for human
ignore.strand	default to TRUE
outputDir	the directory where the off target analysis and reports will be written to
oneFilePergRNA	write to one file for each gRNA or not, default to FALSE
fetchSequence	Fetch flank sequence of off target or not, default TRUE
upstream	upstream offset from the off target start, default 200
downstream	downstream offset from the off target end, default 200
BSgenomeName	BSgenome object. Please refer to available.genomes in BSgenome package. For example, BSgenome.Hsapiens.UCSC.hg19 for hg19, BSgenome.Mmusculus.UCSC.mm10 for mm10, BSgenome.Celegans.UCSC.ce6 for ce6, BSgenome.Rnorvegicus.UCSC.rn5 for rn5, and BSgenome.Dmelanogaster.UCSC.dm3 for dm3
baseBeforegRNA	Number of bases before gRNA used for calculating gRNA efficiency, default 4
baseAfterPAM	Number of bases after PAM used for calculating gRNA efficiency, default 3
featureWeightMatrixFile	Feature weight matrix file used for calculating gRNA efficiency. By default DoenchNBT2014 weight matrix is used. To use alternative weight matrix file, please input a csv file with first column containing significant features and the second column containing the corresponding weights for the features. Please see Doench et al., 2014 for details.
rule.set	Specify a rule set scoring system for calculating gRNA efficacy.
calculategRNAefficacyForOfftargets	Default to TRUE to output gRNA efficacy for offtargets as well as ontargets. Set it to FALSE if only need gRNA efficacy calculated for ontargets only to speed up the analysis. Please refer to https://support.bioconductor.org/p/133538/#133661 for potential use cases of offtarget efficacies.

Value

offtargets	a data frame with off target analysis results
summary	a data frame with summary of the off target analysis results

Author(s)

Lihua Julie Zhu

References

Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol. 2014 Sep 3. doi: 10.1038 nbt.3026 Lihua Julie Zhu, Benjamin R. Holmes, Neil Aronin and Michael Brodsky. CRISPRseek: a Bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. Plos One Sept 23rd 2014

See Also

offTargetAnalysis

Examples

    library(CRISPRseek)
    library("BSgenome.Hsapiens.UCSC.hg19")
    library(TxDb.Hsapiens.UCSC.hg19.knownGene)
    library(org.Hs.eg.db)
    hitsFile <-  system.file("extdata", "hits.txt", package="CRISPRseek")
    hits <- read.table(hitsFile, sep = "\t", header = TRUE, 
        stringsAsFactors = FALSE)
    featureVectors <- buildFeatureVectorForScoring(hits)
    scores <- getOfftargetScore(featureVectors)
    outputDir <- getwd() 
    results <- filterOffTarget(scores, BSgenomeName = Hsapiens, 
        txdb = TxDb.Hsapiens.UCSC.hg19.knownGene,
         orgAnn = org.Hs.egSYMBOL, outputDir = outputDir, 
        min.score = 0.1, topN = 10, topN.OfftargetTotalScore = 5)
    results$offtargets
    results$summary

CRISPRseek documentation built on Jan. 14, 2021, 2:50 a.m.