Hi Julie,

I am using custom parameter values for the individual steps which I am trying to use in the workflow version as well. However, I am getting an error:

Empty hits!Done with offtarget search!
Error in GUIDEseqAnalysis(alignment.inputfile = bamfile, umi.inputfile = umifile,  : 
  No offtargets found with the searching criteria!
In addition: There were 50 or more warnings (use warnings() to see the first 50)

The individual steps are:

uniqueCleavageEvents <- getUniqueCleavageEvents(bamfile, umifile, n.cores.max =4,min.mapping.quality = 15,max.R1.len = 150, max.R2.len =150) #min.R1.mapped = 30,min.R2.mapped=30)

peaks <- getPeaks(uniqueCleavageEvents$cleavage.gr, min.reads = 2,min.SNratio = 2, maxP =0.01,window.size = 25,step = 25 ) #,window.size = 25L,step = 25L
peaks.gr <- peaks$peaks


mergedPeaks <- mergePlusMinusPeaks(peaks.gr = peaks.gr, output.bedfile = bedfile,plus.strand.start.gt.minus.strand.end = FALSE,distance.threshold = 1000) #300


outputDir <-  paste0(name,"_guideseq_out")
dir.create(outputDir)
offTargets <- offTargetAnalysisOfPeakRegions(gRNA = gRNAs, peaks = bedfile,
                                               format=c("fasta", "bed"),
                                               peaks.withHeader = FALSE, BSgenomeName = Hsapiens,
                                               upstream = 50, downstream =50, PAM.size = 3, gRNA.size = 20,
                                               PAM = "NGG", PAM.pattern = "(NGG)$", max.mismatch = 6,
                                               outputDir = outputDir,
                                               orderOfftargetsBy = "predicted_cleavage_score",
                                               allowed.mismatch.PAM = 2, overwrite = TRUE
                                               )

Using the same parameters in the workflow:

GUIDEseqAnalysis(alignment.inputfile = bamfile , umi.inputfile = umifile,
                        alignment.format = c("bam"),
                        BSgenomeName = Hsapiens,
                        gRNA.file = gRNAs,n.cores.max =4,min.mapping.quality = 15,max.R1.len = 150, max.R2.len =150,
                        min.reads = 2,min.SNratio = 2, maxP =0.01,window.size = 25,step = 25,
                        plus.strand.start.gt.minus.strand.end = FALSE,distance.threshold = 1000,
                        upstream = 50, downstream =50, PAM.size = 3, gRNA.size = 20,
                        PAM = "NGG", PAM.pattern = "(NGG)$", max.mismatch = 6,
                        outputDir = outputDir,
                        orderOfftargetsBy = "predicted_cleavage_score",
                        allowed.mismatch.PAM = 2, overwrite = TRUE)

Can you please help with this error?

Thanks Sharvari

Hi Sharvari,

Message " No offtargets found with the searching criteria!" means that your searching criteria is too stringent. You could tune parameters such as max.mismatch = 6, PAM.pattern = "NNN$", upstream = 60, downstream = 60.

Can you check whether the bedfile generated in two ways are the same?

FYI, peak_score column should contain the read count for each offtarget.

Best, Julie