Package: offtracker
OFF-TRACKEROFF-TRACKER is an end to end pipeline of Track-seq data analysis for detecting off-target sites of any genome editing tools that generate double-strand breaks (DSBs) or single-strand breaks (SSBs).System requirementsLinux/UnixPython >= 3.6Dependency# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems# If you don't use mamba, just replace the code with condamambacreate-nofftracker-cbiocondablastsnakemakepybedtoolsInstallation# activate the environmentcondaactivateofftracker# Direct installation with pippipinstallofftracker# (Alternative) Download the offtracker from githubgitclonehttps://github.com/Lan-lab/offtracker.gitcdofftracker
pipinstall.Before analyzing samples# Build blast index (only need once for each genome)makeblastdb-input_typefasta-titlehg38-dbtypenucl-parse_seqids\-in/Your_Path_To_Reference/hg38_genome.fa\-out/Your_Path_To_Reference/hg38_genome.blastdb\-logfile/Your_Path_To_Reference/hg38_genome.blastdb.log# Build chromap index (only need once for each genome)chromap-i-r/Your_Path_To_Reference/hg38_genome.fa\-o/Your_Path_To_Reference/hg38_genome.chromap.index# Generate candidate regions by sgRNA sequence (need once for each genome and sgRNA)offtracker_candidates.py-t8-ghg38\-r/Your_Path_To_Reference/hg38_genome.fa\-b/Your_Path_To_Reference/hg38_genome.blastdb\--name'HEK4'--sgrna'GGCACTGCGGCTGGAGGTGG'--pam'NGG'\-o/Your_Path_To_CandidatesStrand-specific mapping of Track-seq data# Generate snakemake config fileofftracker_config.py-t8-ghg38--blacklisthg38\-r/Your_Path_To_Reference/hg38_genome.fa\-i/Your_Path_To_Reference/hg38_genome.chromap.index\-f/Your_Path_To_Fastq\-o/Your_Path_To_Output\--subfolder0# --subfolder: If different samples are in seperate folders, set this to 1# -o: Default is outputting to /Your_Path_To_Fastq# Run the snakemake programcd/Your_Path_To_Fastq
snakemake-np# dry runnohupsnakemake--cores161>snakemake.log2>snakemake.err&## about cores# --cores of snakemake must be larger than -t of offtracker_config.py# parallel number = cores/t## about output# This part will generate "*.fw.scaled.bw" and ".rv.scaled.bw" for IGV visualization# "*.fw.bed" and "*.rv.bed" are used in the next part.Analyzing the off-target sites# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample namesofftracker_analysis.py-ghg38--name"HEK4"\--exp'Cas9_HEK4.*293'\--control'control'\--outname'Cas9_HEK4_293'\-f/Your_Path_To_Output\--seqfolder/Your_Path_To_Candidates# --name: the same as that in offtracker_candidates.py# --exp/--control: add one or multiple patterns of file name in regex# This step will generate Trackseq_result_{outname}.csv# Intermediate files are saved in ./temp folder, which can be deleted# Keeping the intermediate files can make the analysis faster if involving previously analyzed samples (e.g. using the same control samples for different analyses)Note1The default setting only includes chr1-chr22, chrX, chrY, and chrM.Please make sure the reference genome contains "chr" at the beginning.If you have requirement for other chromosomes or species other than human/mouse, please post an issue.Note2Currently, this software is only ready-to-use for mm10 and hg38.For any other genome, say hg19, please add genome size file named "hg19.chrom.sizes" to .\offtracker\mapping before install.Besides, add "--blacklist none" or "--blacklist Your_Blacklist" when running offtracker_config.pyNote3The FDR in the Track-seq result is not rigorous to the real off-target probability.
It is strongly recommended to observe the "fw.scaled.bw" and "rv.scaled.bw" using IGV to check each target location from the Track-seq result.