Hi Julie,

Thanks for the help.

I get the below error:

Error in read.table(peaks, sep = "\t", header = peaks.withHeader, stringsAsFactors = FALSE) : no lines available in input In addition: Warning message: In file(file, "rt") : file("") only supports open = "w+" and open = "w+b": using the former

My bed file:

head(read.table("./i505_i706.sort.bed")) V1 V2 V3 1 chr1 10865 10902 2 chr1 10960 10999 3 chr1 12855 12947 4 chr1 12915 13008 5 chr1 20261 20296 6 chr1 20412 20447

Input:

library("BSgenome.Hsapiens.UCSC.hg38") library(TxDb.Hsapiens.UCSC.hg38.knownGene) library(org.Hs.eg.db) peaks <- system.file("extdata", "i505i706.sort.bed", package = "CRISPRseek") gRNAs <- system.file("extdata", "SpyDTS4.fa", package = "CRISPRseek") outputDir = getwd() offTargets <- offTargetAnalysisOfPeakRegions(gRNA = gRNAs, peaks = peaks, format=c("fasta", "bed"), peaks.withHeader = FALSE, BSgenomeName = Hsapiens, upstream = 20L, downstream = 20L, PAM.size = 3L, gRNA.size = 20L, orderOfftargetsBy = "predictedcleavagescore", PAM = "NGG", PAM.pattern = "(NGG|NAG|NGA)$", max.mismatch = 2L, outputDir = outputDir, allowed.mismatch.PAM = 3, overwrite = TRUE)

Solution from Julie Zhu

Guideseq cannot handle BED3 format (chr start stop). There must be at-least 4 columns. Column 4 is a unique peak name (see below).

chr1 10014 10057 chr1CWw9JAlsPUc

Hi Tom,

Please set peaks and gRNAs to the file path of your bed file and gRNAs fasta file respectively.

library("BSgenome.Hsapiens.UCSC.hg38")

library(TxDb.Hsapiens.UCSC.hg38.knownGene)

library(org.Hs.eg.db)

peaks <- "i505i706.sort.bed"

gRNAs <- "SpyDTS4.fa"

outputDir = getwd()

offTargets <- offTargetAnalysisOfPeakRegions(gRNA = gRNAs, peaks = peaks,
    format=c("fasta", "bed"),
    peaks.withHeader = TRUE, BSgenomeName = Hsapiens,
    upstream = 20L, downstream = 20L, PAM.size = 3L, gRNA.size = 20L,
    orderOfftargetsBy = "predicted_cleavage_score",
    PAM = "NGG", PAM.pattern = "(NGG|NAG|NGA)$", max.mismatch = 2L,
    outputDir = outputDir,
    allowed.mismatch.PAM = 3, overwrite = TRUE)
annotatedOfftargets <- annotateOffTargets(offTargets,
   txdb = TxDb.Hsapiens.UCSC.hg19.knownGene,
   orgAnn = org.Hs.egSYMBOL)

Best, Julie