Xingjie, What is the goal of your study? Do you need to filter gRNAs using off target score or gRNA efficacy? To obtain all the gRNAs for a given genome without any filtering criteria, you can use the following script. Please change the genome, directory and library location accordingly. Best regards, Julie ######### gRNAsearch-wholeGenHg38.R library(CRISPRseek) library(BSgenome.Hsapiens.UCSC.hg38.masked) args=commandArgs(trailingOnly = TRUE) setwd("~/project/NAD/hg38") chrom <- args[1] batch.ind <- as.numeric(args[2]) chrom.size <- length(Hsapiens[[chrom]]) #### batch.ind 0 - 4 ######### search in main chromosome only ######## gRNAPlusPAM.size <- 23 max.b <- ceiling(chrom.size / 1000000) extra.bases2search <- (gRNAPlusPAM.size - 1) * (max.b - 1) ##### run in 5 jobs max.b <- ceiling((chrom.size + extra.bases2search) / 1000000) batch.start.bin <- min(batch.ind * ceiling(max.b/5), max.b) batch.end.bin <- min(batch.start.bin + ceiling(max.b/5) - 1 , max.b) for (i in batch.start.bin:batch.end.bin) { start <- min(i * 1000000 + 1 - i * (gRNAPlusPAM.size - 1), chrom.size) end <- min(start + 1000000 - 1 , chrom.size) if(i == max.b) end <- chrom.size inputFilePath <- DNAStringSet(substr(Hsapiens[[chrom]], start, end)) names(inputFilePath) <- paste(chrom, start, end, sep = "-") outputDir <- paste("hg38gRNAs", chrom, "_", start, "_", end, sep = "") print(inputFilePath) flag <- TRUE tryCatch(results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, findPairedgRNAOnly = FALSE, annotateExon = FALSE, fetchSequence = FALSE, annotatePaired = FALSE, gRNAoutputName = "hg38", findgRNAs = TRUE,exportAllgRNAs = "fasta", enable.multicore = TRUE, n.cores.max = 20, BSgenomeName = Hsapiens, chromToSearch = "", max.mismatch = 0, PAM.pattern = "NGG$", outputDir = outputDir, overwrite = TRUE), error=function(e) flag<<-FALSE) if (!flag) next } } Here is the batch script for running with bsub. Please change the genome, directory and library location accordingly. for chrom in chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 ch r19 chr20 chr21 chr22 chrX chrY; do for FILE in $(seq 0 4); do SHF=seq-${chrom}-${FILE}.bsub echo "#!/bin/bash" > $SHF echo "#BSUB -P $FILE" >>$SHF echo "workingDir=/project/CRISPRseek" >>$SHF echo "export R_LIBS=/project/umw_mccb/R/R-3.3.1/lib64/R/library:/share/pkg/R/3.3.1/lib64/R/library :/home/jz57w/R/x86_64-pc-linux-gnu-library/3.3" >>$SHF workingDir=/project/CRISPRseek/hg38 echo "cd $workingDir" >>$SHF echo "#BSUB -q long" >> $SHF echo "#BSUB -R rusage[mem=20000]" >> $SHF echo "#BSUB -W 240:00" >>$SHF echo "#BSUB -o out.${chrom}.${FILE}.log" >>$SHF echo "#BSUB -e err.${chrom}.${FILE}.log" >>$SHF echo "/project/umw_mccb/bin/R CMD BATCH --no-save --no-restore '--args $chrom $FILE' /project/CRISPRseek/hg38/gRNAsearch-wholeGenHg38.R $SHF.log" >> $SHF bsub < $SHF sleep 5 done Done Best, Julie From: "renxj2008 [bioc]" <noreply@bioconductor.org<mailto:noreply@bioconductor.org>> Reply-To: "reply+6d139c94+code@bioconductor.org<mailto:reply+6d139c94+code@bioconductor.org>" <reply+6d139c94+code@bioconductor.org<mailto:reply+6d139c94+code@bioconductor.org>> Date: Friday, April 14, 2017 1:37 AM To: Lihua Julie Zhu <julie.zhu@umassmed.edu<mailto:julie.zhu@umassmed.edu>> Subject: [bioc] tilling sgRNA library design Activity on a post you are following on support.bioconductor.org<https: support.bioconductor.org=""> User renxj2008<https: support.bioconductor.org="" u="" 12842=""/> wrote Question: tilling sgRNA library design<https: support.bioconductor.org="" p="" 94964=""/>: Hi, I found the CRISPRseek is a good platform to design the sgRNA. Is there someone try to use it design the tilling sgRNA? If who know how to design the tilling sgRNA, please tell me at xingjie.ren@ucsf.edu<mailto:xingjie.ren@ucsf.edu>. Thanks very much! Best, xingjie ________________________________ Post tags: bioconductor, crisprseek You may reply via email or visit tilling sgRNA library design