offtracker 2.7.8__zip → 2.7.10__zip

offtracker-2.7.10/PKG-INFO

@@ -0,0 +1,189 @@
+Metadata-Version: 2.1
+Name: offtracker
+Version: 2.7.10
+Summary: Tracking-seq data analysis
+Home-page: https://github.com/Lan-lab/offtracker
+Author: Runda Xu
+Author-email: runda.xu@foxmail.com
+Requires-Python: >=3.6.0
+Description-Content-Type: text/markdown
+License-File: LICENSE.txt
+
+
+# OFF-TRACKER
+
+OFF-TRACKER is an end to end pipeline of Tracking-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 requirements
+
+* Linux/Unix 
+* Python >= 3.6
+
+## Dependency
+
+```bash
+# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems
+# If you don't use mamba, just replace the code with conda 
+mamba create -n offtracker -c bioconda blast snakemake pybedtools
+```
+
+
+## Installation 
+
+```bash
+# Activate the environment
+conda activate offtracker
+
+# Direct installation with pip
+pip install offtracker
+
+# (Alternative) Download the offtracker from github
+git clone https://github.com/Lan-lab/offtracker.git 
+cd offtracker
+pip install .
+```
+
+
+## Before analyzing samples
+
+```bash
+# Build blast index (only need once for each genome)
+makeblastdb -input_type fasta -title hg38 -dbtype nucl -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)
+# --name: the name of the sgRNA, which will be used in the following analysis
+offtracker_candidates.py -t 8 -g hg38 \
+-r /Your_Path_To_Reference/hg38_genome.fa \
+-b /Your_Path_To_Reference/hg38_genome.blastdb \
+--name 'VEGFA2' --sgrna 'GACCCCCTCCACCCCGCCTC' --pam 'NGG' \
+-o /Your_Path_To_Candidates
+
+```
+
+## Strand-specific mapping of Tracking-seq data 
+
+```bash
+# Generate snakemake config file 
+# --subfolder: If different samples are in seperate folders, set this to 1
+# if -o is not set, the output will be in the same folder as the fastq files
+offtracker_config.py -t 8 -g hg38 --blacklist hg38 \
+-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 \ 
+--subfolder 0 
+
+# Run the snakemake program
+cd /Your_Path_To_Fastq
+snakemake -np # dry run
+nohup snakemake --cores 16 1>snakemake.log 2>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 genome-wide off-target sites
+
+```bash
+# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample names
+
+offtracker_analysis.py -g hg38 --name "VEGFA2" \
+--exp 'Cas9_VEGFA2' \
+--control 'WT' \
+--outname 'Cas9_VEGFA_293' \
+-f /Your_Path_To_Output \
+--seqfolder /Your_Path_To_Candidates
+
+# --name: the same gRNA name you set when running offtracker_candidates.py
+# --exp/--control: add one or multiple patterns of file name in regular expressions
+# If multiple samples meet the pattern, their signals will be averaged. Thus, only samples with the same condition should be included in a single analysis.
+
+# This step will generate Offtracker_result_{outname}.csv
+# Default FDR is 0.05, which can be changed by --fdr. This will empirically make the threshold of Track score around 2.
+# Sites with Track score >=2, which is a empirical threshold, are output regardless of FDR.
+# 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)
+```
+
+## Off-target sequences visualization
+
+```bash
+# After get the Offtracker_result_{outname}.csv, you can visualize the off-target sites with their genomic sequence with the following command:
+
+offtracker_plot.py --result Your_Offtracker_Result_CSV \
+--sgrna 'GACCCCCTCCACCCCGCCTC' --pam 'NGG'
+
+# The default output is a pdf file with Offtracker_result_{outname}.pdf
+# Change the suffix of the output file to change the format (e.g.: .png)
+# The orange dash line indicates the empirical threshold of Track score = 2
+# Empirically, the off-target sites with Track score < 2 are less likely to be real off-target sites.
+```
+
+
+## Note1
+
+The default setting only includes chr1-chr22, chrX, chrY, and chrM. Please make sure the reference genome contains "chr" at the beginning. 
+
+Currently, this software is only ready-to-use for mm10 and hg38. For any other genome, e.g., hg19, please add genome size file named "hg19.chrom.sizes" to .\offtracker\mapping and instal manually. Besides, add "--blacklist none" or "--blacklist Your_Blacklist" (e.g., ENCODE blacklist) when running offtracker_config.py, because we only provide blacklists for mm10 and hg38.
+
+If you have a requirement for species other than human/mouse, please post an issue.
+
+## Note2
+
+The FDRs in the Tracking-seq result do not reflect the real off-target probability.
+It is strongly recommended to observe the "fw.scaled.bw" and "rv.scaled.bw" using genome browser like IGV to visually inspect each target location from the Tracking-seq result.
+
+
+
+# Example Data
+
+Here are example data that contains reads of chr6 from HEK293T cells edited with Cas9 + sgRNA VEGFA2 and wild type cells:
+
+https://figshare.com/articles/dataset/WT_HEK239T_chr6/25956034
+
+It takes about 5-10 minutes to run the mapping (offtracker_config.py & snakemake) of example data with -t 8 and --cores 16 (2 parallel tasks)
+
+## Signal visualization
+
+After mapping, there will be 4 .bw files in the output folder:
+```bash
+Cas9_VEGFA2_chr6.fw.scaled.bw
+
+Cas9_VEGFA2_chr6.rv.scaled.bw
+
+WT_chr6.fw.scaled.bw
+
+WT_chr6.rv.scaled.bw
+```
+These files can be visualized in genome browser like IGV:
+
+![signal](https://github.com/Lan-lab/offtracker/blob/main/example_output/signals_example.png?raw=true)
+
+
+## Whole genome off-target analysis
+
+For analyzing the signals (offtracker_analysis.py), it takes about 3-5 minutes and outputs a file named "Offtracker_result_{outname}.csv"
+
+After that, you can visualize the off-target sites with their genomic sequence (offtracker_plot.py) and get an image like this:
+
+![offtarget](https://github.com/Lan-lab/offtracker/blob/main/example_output/sequences_example.png?raw=true)
+
+# Citation
+
+
+
+
+

offtracker-2.7.10/README.md

@@ -0,0 +1,177 @@
+# OFF-TRACKER
+
+OFF-TRACKER is an end to end pipeline of Tracking-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 requirements
+
+* Linux/Unix 
+* Python >= 3.6
+
+## Dependency
+
+```bash
+# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems
+# If you don't use mamba, just replace the code with conda 
+mamba create -n offtracker -c bioconda blast snakemake pybedtools
+```
+
+
+## Installation 
+
+```bash
+# Activate the environment
+conda activate offtracker
+
+# Direct installation with pip
+pip install offtracker
+
+# (Alternative) Download the offtracker from github
+git clone https://github.com/Lan-lab/offtracker.git 
+cd offtracker
+pip install .
+```
+
+
+## Before analyzing samples
+
+```bash
+# Build blast index (only need once for each genome)
+makeblastdb -input_type fasta -title hg38 -dbtype nucl -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)
+# --name: the name of the sgRNA, which will be used in the following analysis
+offtracker_candidates.py -t 8 -g hg38 \
+-r /Your_Path_To_Reference/hg38_genome.fa \
+-b /Your_Path_To_Reference/hg38_genome.blastdb \
+--name 'VEGFA2' --sgrna 'GACCCCCTCCACCCCGCCTC' --pam 'NGG' \
+-o /Your_Path_To_Candidates
+
+```
+
+## Strand-specific mapping of Tracking-seq data 
+
+```bash
+# Generate snakemake config file 
+# --subfolder: If different samples are in seperate folders, set this to 1
+# if -o is not set, the output will be in the same folder as the fastq files
+offtracker_config.py -t 8 -g hg38 --blacklist hg38 \
+-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 \ 
+--subfolder 0 
+
+# Run the snakemake program
+cd /Your_Path_To_Fastq
+snakemake -np # dry run
+nohup snakemake --cores 16 1>snakemake.log 2>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 genome-wide off-target sites
+
+```bash
+# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample names
+
+offtracker_analysis.py -g hg38 --name "VEGFA2" \
+--exp 'Cas9_VEGFA2' \
+--control 'WT' \
+--outname 'Cas9_VEGFA_293' \
+-f /Your_Path_To_Output \
+--seqfolder /Your_Path_To_Candidates
+
+# --name: the same gRNA name you set when running offtracker_candidates.py
+# --exp/--control: add one or multiple patterns of file name in regular expressions
+# If multiple samples meet the pattern, their signals will be averaged. Thus, only samples with the same condition should be included in a single analysis.
+
+# This step will generate Offtracker_result_{outname}.csv
+# Default FDR is 0.05, which can be changed by --fdr. This will empirically make the threshold of Track score around 2.
+# Sites with Track score >=2, which is a empirical threshold, are output regardless of FDR.
+# 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)
+```
+
+## Off-target sequences visualization
+
+```bash
+# After get the Offtracker_result_{outname}.csv, you can visualize the off-target sites with their genomic sequence with the following command:
+
+offtracker_plot.py --result Your_Offtracker_Result_CSV \
+--sgrna 'GACCCCCTCCACCCCGCCTC' --pam 'NGG'
+
+# The default output is a pdf file with Offtracker_result_{outname}.pdf
+# Change the suffix of the output file to change the format (e.g.: .png)
+# The orange dash line indicates the empirical threshold of Track score = 2
+# Empirically, the off-target sites with Track score < 2 are less likely to be real off-target sites.
+```
+
+
+## Note1
+
+The default setting only includes chr1-chr22, chrX, chrY, and chrM. Please make sure the reference genome contains "chr" at the beginning. 
+
+Currently, this software is only ready-to-use for mm10 and hg38. For any other genome, e.g., hg19, please add genome size file named "hg19.chrom.sizes" to .\offtracker\mapping and instal manually. Besides, add "--blacklist none" or "--blacklist Your_Blacklist" (e.g., ENCODE blacklist) when running offtracker_config.py, because we only provide blacklists for mm10 and hg38.
+
+If you have a requirement for species other than human/mouse, please post an issue.
+
+## Note2
+
+The FDRs in the Tracking-seq result do not reflect the real off-target probability.
+It is strongly recommended to observe the "fw.scaled.bw" and "rv.scaled.bw" using genome browser like IGV to visually inspect each target location from the Tracking-seq result.
+
+
+
+# Example Data
+
+Here are example data that contains reads of chr6 from HEK293T cells edited with Cas9 + sgRNA VEGFA2 and wild type cells:
+
+https://figshare.com/articles/dataset/WT_HEK239T_chr6/25956034
+
+It takes about 5-10 minutes to run the mapping (offtracker_config.py & snakemake) of example data with -t 8 and --cores 16 (2 parallel tasks)
+
+## Signal visualization
+
+After mapping, there will be 4 .bw files in the output folder:
+```bash
+Cas9_VEGFA2_chr6.fw.scaled.bw
+
+Cas9_VEGFA2_chr6.rv.scaled.bw
+
+WT_chr6.fw.scaled.bw
+
+WT_chr6.rv.scaled.bw
+```
+These files can be visualized in genome browser like IGV:
+
+![signal](https://github.com/Lan-lab/offtracker/blob/main/example_output/signals_example.png?raw=true)
+
+
+## Whole genome off-target analysis
+
+For analyzing the signals (offtracker_analysis.py), it takes about 3-5 minutes and outputs a file named "Offtracker_result_{outname}.csv"
+
+After that, you can visualize the off-target sites with their genomic sequence (offtracker_plot.py) and get an image like this:
+
+![offtarget](https://github.com/Lan-lab/offtracker/blob/main/example_output/sequences_example.png?raw=true)
+
+# Citation
+
+
+
+
+

{offtracker-2.7.8 → offtracker-2.7.10}/offtracker/X_offplot.py

@@ -1,13 +1,22 @@
-import matplotlib.pyplot as plt
-import matplotlib.patches as patches
+
 import pandas as pd
 import numpy as np
-
-def offtable(offtargets, target_guide, 
+import matplotlib.pyplot as plt
+import matplotlib.patches as patches
+from matplotlib import rcParams
+# 和用 plt.rcParams or matplotlib.rcParams 是一样的
+dict_rc = {
+    'pdf.fonttype': 42,
+    'font.family': ['Arial']
+}
+rcParams.update(dict_rc)
+
+# 2024.06.03. offtable 添加 threshold 分界线，默认为 None，常用的是 2
+def offtable(offtargets, target_guide,  length_pam = 3, 
                          col_seq='best_target', col_score='track_score', col_mismatch='mismatch', col_loc='target_location',
                          title=None, font='Arial', font_size=9, 
-                         box_size_x=15, box_size_y=20, box_gap=1,
-                         x_offset=15, y_offset=35, dpi=200, savefig=None):
+                         box_size_x=15, box_size_y=20, box_gap=1, threshold=None,
+                         x_offset=15, y_offset=35, dpi=300, savefig=None):
     # Facecolor
     color_dict = {
         'A': 'lightgreen',
@@ -21,6 +30,8 @@ def offtable(offtargets, target_guide,
 
     # If offtargets is a DataFrame, convert to list of dictionaries
     if isinstance(offtargets, pd.DataFrame):
+        if threshold is not None:
+            n_positive = sum(offtargets[col_score]>=threshold)
         offtargets = offtargets.to_dict(orient='records')
 
     # Configuration
@@ -100,11 +111,18 @@ def offtable(offtargets, target_guide,
         ax.text(x_offset + (len(target_guide) + 4) * box_size_x, y + box_size_y / 2, seq[col_loc], ha='left', va='center', family=font, fontsize=font_size)
 
     # add a vertical line to indicate the PAM
-    x_line = x_offset + (len(target_guide) - 3) * box_size_x
+    x_line = x_offset + (len(target_guide) - length_pam) * box_size_x
     y_start = y_offset # + box_size_y / 2 
     y_end = y_start + (len(offtargets)+1) * (box_size_y + box_gap)
     ax.vlines(x=x_line, ymin=y_start, ymax=y_end, color='indianred', linestyle='--')
 
+    # 2024.06.03. add a horizontal line to indicate the threshold
+    if threshold is not None:
+        thresh_x_start = x_offset
+        thresh_x_end = x_offset + len(target_guide) * box_size_x
+        thresh_y = y_offset + (n_positive+1) * (box_size_y + box_gap) - box_gap*0.5
+        ax.hlines(y=thresh_y, xmin=thresh_x_start, xmax=thresh_x_end, color='orange', linestyle='--')
+
     # Styling and save
     ax.set_xlim(0, width*1.1) # location 的文字太长了，所以要加长一点
     ax.set_ylim(height, 0)

offtracker-2.7.10/offtracker/_version.py

@@ -0,0 +1,30 @@
+__version__ = "2.7.10"
+# 2023.08.11. v1.1.0	adding a option for not normalizing the bw file
+# 2023.10.26. v1.9.0	prerelease for v2.0
+# 2023.10.27. v2.0.0	大更新，还没微调
+# 2023.10.28. v2.1.0	修复bug，增加计算信号长度的功能
+# 2023.10.28. v2.2.0	修复bug，改变计算信号长度的算法
+# 2023.10.29. v2.3.0	增加 overall signal 计算
+# 2023.11.01. v2.3.1	增加 signal_only 选项
+# 2023.11.02. v2.3.2	修改 sample signal 和 group mean 的计算顺序
+# 2023.11.04. v2.3.3	修复 overall score 标准化时排序错误的问题
+# 2023.11.05. v2.3.4	修复判断单边溢出信号时的列名选取错误
+# 2023.11.13. v2.3.5	微调 track score
+# 2023.12.05. v2.3.6	candidates 增加 cleavage site，修正 alignment 有 deletion 会错位的 bug
+# 2023.12.05. v2.3.7	用 cleavage site 代替 midpoint # 还没改完
+# 2023.12.07. v2.3.8	df_score 增加 df_exp, df_ctr 各自列。修复没 df_ctr 时的 bug。track score 用 proximal
+# 2023.12.09. v2.4.0	为了兼顾 proximal 和 overall，当 normalized overall signal 高于 2 时，增加 overall signal 的加分
+# 2023.12.09. v2.5.0	尝试新的加权位置
+# 2023.12.10. v2.6.0	加入 trackseq v4 的计算分支，即考虑 Region 内的 positive_pct，避免短而尖锐的信号
+# 2023.12.10. v2.6.1	有些非特异信号数值很大，如果在 control 组是大负数，可能导致减 control 后假高信号，因此给负数一个 clip
+# 2023.12.30. v2.7.0	增加 X_offplot 模块，用于绘图
+# 2023.12.31. v2.7.1	control 的负数值 clip 由 -5 改为 -1，进一步减少假阳性。另外不加 overall 了
+# 2024.01.01. v2.7.2	权重改为 proximal + pct = 1 + 1. 防信号外溢假阳性标准由<0改为<=0
+# 2024.01.02. v2.7.3	flank regions 默认值改为 1000 2000 3000 5000。之前 control 的负数值 clip 相当于直接在 final score，现在改为每个单独 clip 后重新算 score，默认值为 CtrClip=-0.5
+# 2024.01.03. v2.7.4	更新了 blacklist.bed
+# 2024.01.04. v2.7.5	更新了 hg38 blacklist.bed
+# 2024.01.12. v2.7.6	修复小bug，输出 fdr 改为 <0.05。
+# 2024.01.23. v2.7.7	Snakefile_offtracker: add --fixedStep to bigwigCompare for not merging neighbouring bins with equal values.
+# 2024.02.01. v2.7.8	逐步添加 X_offplot.py 功能
+# 2024.06.02. v2.7.9	添加 offtracker_plot.py
+# 2024.06.03. v2.7.10	修复 bugs，offtable 添加 threshold = 2 的分界

offtracker-2.7.10/offtracker.egg-info/PKG-INFO

@@ -0,0 +1,189 @@
+Metadata-Version: 2.1
+Name: offtracker
+Version: 2.7.10
+Summary: Tracking-seq data analysis
+Home-page: https://github.com/Lan-lab/offtracker
+Author: Runda Xu
+Author-email: runda.xu@foxmail.com
+Requires-Python: >=3.6.0
+Description-Content-Type: text/markdown
+License-File: LICENSE.txt
+
+
+# OFF-TRACKER
+
+OFF-TRACKER is an end to end pipeline of Tracking-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 requirements
+
+* Linux/Unix 
+* Python >= 3.6
+
+## Dependency
+
+```bash
+# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems
+# If you don't use mamba, just replace the code with conda 
+mamba create -n offtracker -c bioconda blast snakemake pybedtools
+```
+
+
+## Installation 
+
+```bash
+# Activate the environment
+conda activate offtracker
+
+# Direct installation with pip
+pip install offtracker
+
+# (Alternative) Download the offtracker from github
+git clone https://github.com/Lan-lab/offtracker.git 
+cd offtracker
+pip install .
+```
+
+
+## Before analyzing samples
+
+```bash
+# Build blast index (only need once for each genome)
+makeblastdb -input_type fasta -title hg38 -dbtype nucl -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)
+# --name: the name of the sgRNA, which will be used in the following analysis
+offtracker_candidates.py -t 8 -g hg38 \
+-r /Your_Path_To_Reference/hg38_genome.fa \
+-b /Your_Path_To_Reference/hg38_genome.blastdb \
+--name 'VEGFA2' --sgrna 'GACCCCCTCCACCCCGCCTC' --pam 'NGG' \
+-o /Your_Path_To_Candidates
+
+```
+
+## Strand-specific mapping of Tracking-seq data 
+
+```bash
+# Generate snakemake config file 
+# --subfolder: If different samples are in seperate folders, set this to 1
+# if -o is not set, the output will be in the same folder as the fastq files
+offtracker_config.py -t 8 -g hg38 --blacklist hg38 \
+-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 \ 
+--subfolder 0 
+
+# Run the snakemake program
+cd /Your_Path_To_Fastq
+snakemake -np # dry run
+nohup snakemake --cores 16 1>snakemake.log 2>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 genome-wide off-target sites
+
+```bash
+# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample names
+
+offtracker_analysis.py -g hg38 --name "VEGFA2" \
+--exp 'Cas9_VEGFA2' \
+--control 'WT' \
+--outname 'Cas9_VEGFA_293' \
+-f /Your_Path_To_Output \
+--seqfolder /Your_Path_To_Candidates
+
+# --name: the same gRNA name you set when running offtracker_candidates.py
+# --exp/--control: add one or multiple patterns of file name in regular expressions
+# If multiple samples meet the pattern, their signals will be averaged. Thus, only samples with the same condition should be included in a single analysis.
+
+# This step will generate Offtracker_result_{outname}.csv
+# Default FDR is 0.05, which can be changed by --fdr. This will empirically make the threshold of Track score around 2.
+# Sites with Track score >=2, which is a empirical threshold, are output regardless of FDR.
+# 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)
+```
+
+## Off-target sequences visualization
+
+```bash
+# After get the Offtracker_result_{outname}.csv, you can visualize the off-target sites with their genomic sequence with the following command:
+
+offtracker_plot.py --result Your_Offtracker_Result_CSV \
+--sgrna 'GACCCCCTCCACCCCGCCTC' --pam 'NGG'
+
+# The default output is a pdf file with Offtracker_result_{outname}.pdf
+# Change the suffix of the output file to change the format (e.g.: .png)
+# The orange dash line indicates the empirical threshold of Track score = 2
+# Empirically, the off-target sites with Track score < 2 are less likely to be real off-target sites.
+```
+
+
+## Note1
+
+The default setting only includes chr1-chr22, chrX, chrY, and chrM. Please make sure the reference genome contains "chr" at the beginning. 
+
+Currently, this software is only ready-to-use for mm10 and hg38. For any other genome, e.g., hg19, please add genome size file named "hg19.chrom.sizes" to .\offtracker\mapping and instal manually. Besides, add "--blacklist none" or "--blacklist Your_Blacklist" (e.g., ENCODE blacklist) when running offtracker_config.py, because we only provide blacklists for mm10 and hg38.
+
+If you have a requirement for species other than human/mouse, please post an issue.
+
+## Note2
+
+The FDRs in the Tracking-seq result do not reflect the real off-target probability.
+It is strongly recommended to observe the "fw.scaled.bw" and "rv.scaled.bw" using genome browser like IGV to visually inspect each target location from the Tracking-seq result.
+
+
+
+# Example Data
+
+Here are example data that contains reads of chr6 from HEK293T cells edited with Cas9 + sgRNA VEGFA2 and wild type cells:
+
+https://figshare.com/articles/dataset/WT_HEK239T_chr6/25956034
+
+It takes about 5-10 minutes to run the mapping (offtracker_config.py & snakemake) of example data with -t 8 and --cores 16 (2 parallel tasks)
+
+## Signal visualization
+
+After mapping, there will be 4 .bw files in the output folder:
+```bash
+Cas9_VEGFA2_chr6.fw.scaled.bw
+
+Cas9_VEGFA2_chr6.rv.scaled.bw
+
+WT_chr6.fw.scaled.bw
+
+WT_chr6.rv.scaled.bw
+```
+These files can be visualized in genome browser like IGV:
+
+![signal](https://github.com/Lan-lab/offtracker/blob/main/example_output/signals_example.png?raw=true)
+
+
+## Whole genome off-target analysis
+
+For analyzing the signals (offtracker_analysis.py), it takes about 3-5 minutes and outputs a file named "Offtracker_result_{outname}.csv"
+
+After that, you can visualize the off-target sites with their genomic sequence (offtracker_plot.py) and get an image like this:
+
+![offtarget](https://github.com/Lan-lab/offtracker/blob/main/example_output/sequences_example.png?raw=true)
+
+# Citation
+
+
+
+
+

{offtracker-2.7.8 → offtracker-2.7.10}/offtracker.egg-info/SOURCES.txt

@@ -22,4 +22,5 @@ offtracker/mapping/offtracker_blacklist_hg38.merged.bed
 offtracker/mapping/offtracker_blacklist_mm10.merged.bed
 scripts/offtracker_analysis.py
 scripts/offtracker_candidates.py
-scripts/offtracker_config.py
+scripts/offtracker_config.py
+scripts/offtracker_plot.py

{offtracker-2.7.8 → offtracker-2.7.10}/scripts/offtracker_analysis.py

@@ -26,6 +26,7 @@ def main():
     parser.add_argument('--name'         , type=str, required=True,    help='custom name of the sgRNA' )
     parser.add_argument('--exp'          , type=str, default='all',    nargs='+', help='A substring mark in the name of experimental samples. The default is to use all samples other than control' )
     parser.add_argument('--control'      , type=str, default='none',   nargs='+', help='A substring mark in the name of control samples. The default is no control. "others" for all samples other than --exp.' )
+    parser.add_argument('--fdr'          , type=int, default=0.05,     help='FDR threshold for the final result. Default is 0.05.')
     parser.add_argument('--smooth'       , type=int, default=1,        help='Smooth strength for the signal.')
     parser.add_argument('--window'       , type=int, default=3,        help='Window size for smoothing the signal.')
     parser.add_argument('--binsize'      , type=int, default=100,      help='Window size for smoothing the signal.')
@@ -49,6 +50,7 @@ def main():
     sgRNA_name = args.name
     pattern_exp = args.exp
     pattern_ctr = args.control
+    fdr_thresh = args.fdr
     binsize = args.binsize
     flank_max = args.flank_max
     flank_regions = args.flank_regions
@@ -155,8 +157,11 @@ def main():
         df_bdg.columns = ['chr','start','end','residual']
         # 将 df_bdg 按照染色体分组
         sample_groups = df_bdg.groupby('chr')
+        # 2024.06.03. fix a bug that df_bdg has less chr than df_candidate
+        total_chr = df_bdg['chr'].unique()
+        df_candidate_sub_temp = df_candidate_sub[df_candidate_sub['chr'].isin(total_chr)]
         # 将 df_candidate_sub 按照染色体分组
-        candidate_groups = df_candidate_sub.groupby('chr')
+        candidate_groups = df_candidate_sub_temp.groupby('chr')
 
         # 定义一个空的列表，用于存储每个染色体的数据
         chrom_list = []
@@ -234,7 +239,8 @@ def main():
             df_score = pd.concat([df_score, df_exp, df_ctr], axis=1)
         else:
             df_score = pd.concat([df_score, df_exp], axis=1)
-        df_score = df_score.copy()
+        # 2024.06.03. 跑样例数据时，只有一个 chr6, 其他都是 nan, 不删除会导致后续计算出错
+        df_score = df_score.dropna().copy()
         df_score.to_csv(output)
     
     ##########################
@@ -339,8 +345,10 @@ def main():
         df_result['fdr'] = offtracker.fdr(df_result['pv'])
         df_result['rank'] = range(1,len(df_result)+1)
         df_result.to_csv(output)        
-
-        df_output = df_result[df_result['fdr']<=0.05].copy()
+        # 2024.06.03. 以防 fdr<=fdr_thresh 滤掉了 track_score>=2 的位点
+        bool_fdr = df_result['fdr']<=fdr_thresh
+        bool_score = df_result['track_score']>=2
+        df_output = df_result[bool_fdr|bool_score].copy()
         if pattern_ctr != 'none':
             df_output = df_output[['target_location', 'best_strand','best_target','deletion','insertion','mismatch',
                                    'exp_L_length', 'exp_R_length','ctr_L_length','ctr_R_length','L_length','R_length','signal_length',

offtracker-2.7.10/scripts/offtracker_plot.py

@@ -0,0 +1,39 @@
+#!/usr/bin/env python
+# -*- coding: utf-8 -*-
+
+import offtracker.X_offplot as xoffplot
+import pandas as pd
+import argparse
+import os
+
+def main():
+    parser = argparse.ArgumentParser()
+    parser.description='Draw the plot of the off-targets with genomic sequences.\nIf .pdf file is too large, try to use .png file instead.'
+    parser.add_argument('--result' , type=str, required=True,  help='The file of Offtracker_result_{outname}.csv' )
+    parser.add_argument('--sgrna'  , type=str, required=True,  help='Not including PAM' )
+    parser.add_argument('--pam'    , type=str, default='NGG',  help='PAM sequence. Default is "NGG".' )
+    parser.add_argument('--output' , type=str, default='same', help='The output file. Default is Offtracker_result_{outname}.pdf')
+
+    args = parser.parse_args()
+    if args.output == 'same':
+        dir_savefig = args.result.replace('.csv', '.pdf')
+    else:
+        dir_savefig = args.output
+
+    outname = os.path.basename(args.result).replace('Offtracker_result_', '').replace('.csv', '')
+    gRNA = args.sgrna
+    PAM = args.pam
+    full_seq = gRNA + PAM
+    
+    df_result = pd.read_csv(args.result)
+    n_pos = len(df_result)
+
+    xoffplot.offtable(df_result, full_seq, length_pam = len(PAM), col_seq='target', threshold=2,
+                      title=f'{outname} ({n_pos} sites)',
+                      savefig=dir_savefig)
+    
+    return f'The plot is saved as {dir_savefig}'
+
+if __name__ == '__main__' :
+    result = main()
+    print(result)

{offtracker-2.7.8 → offtracker-2.7.10}/setup.py

@@ -49,7 +49,10 @@ setup(
     python_requires=REQUIRES_PYTHON,
     packages=find_packages(),
     package_data={'offtracker': ['mapping/*']},
-    scripts = ['scripts/offtracker_config.py','scripts/offtracker_candidates.py','scripts/offtracker_analysis.py'],
+    scripts = ['scripts/offtracker_config.py',
+               'scripts/offtracker_candidates.py',
+               'scripts/offtracker_analysis.py',
+               'scripts/offtracker_plot.py'],
     install_requires=REQUIRED,
     include_package_data=True
 )

offtracker-2.7.8/PKG-INFO

@@ -1,146 +0,0 @@
-Metadata-Version: 2.1
-Name: offtracker
-Version: 2.7.8
-Summary: Tracking-seq data analysis
-Home-page: https://github.com/Lan-lab/offtracker
-Author: Runda Xu
-Author-email: runda.xu@foxmail.com
-Requires-Python: >=3.6.0
-Description-Content-Type: text/markdown
-License-File: LICENSE.txt
-
-
-OFF-TRACKER
-=======================
-
-OFF-TRACKER is an end to end pipeline of Tracking-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 requirements
------
-* Linux/Unix 
-* Python >= 3.6
-
-Dependency
------
-
-```bash
-# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems
-# If you don't use mamba, just replace the code with conda 
-mamba create -n offtracker -c bioconda blast snakemake pybedtools
-```
-
-
-Installation 
------
-
-```bash
-# activate the environment
-conda activate offtracker
-
-# Direct installation with pip
-pip install offtracker
-
-# (Alternative) Download the offtracker from github
-git clone https://github.com/Lan-lab/offtracker.git 
-cd offtracker
-pip install .
-```
-
-
-Before analyzing samples
------
-
-```bash
-# Build blast index (only need once for each genome)
-makeblastdb -input_type fasta -title hg38 -dbtype nucl -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 -t 8 -g hg38 \
--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_Candidates
-
-```
-
-Strand-specific mapping of Tracking-seq data 
------
-
-```bash
-# Generate snakemake config file 
-offtracker_config.py -t 8 -g hg38 --blacklist hg38 \
--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 \ 
---subfolder 0 
-
-# --subfolder: If different samples are in seperate folders, set this to 1
-# -o: Default is outputting to /Your_Path_To_Fastq
-
-# Run the snakemake program
-cd /Your_Path_To_Fastq
-snakemake -np # dry run
-nohup snakemake --cores 16 1>snakemake.log 2>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
------
-
-```bash
-# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample names
-
-offtracker_analysis.py -g hg38 --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)
-```
-
-
-Note1
---------------
-The 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.
-
-Note2
---------------
-Currently, 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.py
-
-Note3
---------------
-The FDR in the Tracking-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 Tracking-seq result.
-

offtracker-2.7.8/README.md

@@ -1,134 +0,0 @@
-OFF-TRACKER
-=======================
-
-OFF-TRACKER is an end to end pipeline of Tracking-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 requirements
------
-* Linux/Unix 
-* Python >= 3.6
-
-Dependency
------
-
-```bash
-# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems
-# If you don't use mamba, just replace the code with conda 
-mamba create -n offtracker -c bioconda blast snakemake pybedtools
-```
-
-
-Installation 
------
-
-```bash
-# activate the environment
-conda activate offtracker
-
-# Direct installation with pip
-pip install offtracker
-
-# (Alternative) Download the offtracker from github
-git clone https://github.com/Lan-lab/offtracker.git 
-cd offtracker
-pip install .
-```
-
-
-Before analyzing samples
------
-
-```bash
-# Build blast index (only need once for each genome)
-makeblastdb -input_type fasta -title hg38 -dbtype nucl -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 -t 8 -g hg38 \
--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_Candidates
-
-```
-
-Strand-specific mapping of Tracking-seq data 
------
-
-```bash
-# Generate snakemake config file 
-offtracker_config.py -t 8 -g hg38 --blacklist hg38 \
--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 \ 
---subfolder 0 
-
-# --subfolder: If different samples are in seperate folders, set this to 1
-# -o: Default is outputting to /Your_Path_To_Fastq
-
-# Run the snakemake program
-cd /Your_Path_To_Fastq
-snakemake -np # dry run
-nohup snakemake --cores 16 1>snakemake.log 2>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
------
-
-```bash
-# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample names
-
-offtracker_analysis.py -g hg38 --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)
-```
-
-
-Note1
---------------
-The 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.
-
-Note2
---------------
-Currently, 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.py
-
-Note3
---------------
-The FDR in the Tracking-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 Tracking-seq result.
-

offtracker-2.7.8/offtracker/_version.py

@@ -1,28 +0,0 @@
-__version__ = "2.7.8"
-# 2023.08.11. v1.1.0	adding a option for not normalizing the bw file
-# 2023.10.26. v1.9.0	prerelease for v2.0
-# 2023.10.27. v2.0.0	大更新，还没微调
-# 2023.10.28. v2.1.0 修复bug，增加计算信号长度的功能
-# 2023.10.28. v2.2.0 修复bug，改变计算信号长度的算法
-# 2023.10.29. v2.3.0 增加 overall signal 计算
-# 2023.11.01. v2.3.1 增加 signal_only 选项
-# 2023.11.02. v2.3.2 修改 sample signal 和 group mean 的计算顺序
-# 2023.11.04. v2.3.3 修复 overall score 标准化时排序错误的问题
-# 2023.11.05. v2.3.4 修复判断单边溢出信号时的列名选取错误
-# 2023.11.13. v2.3.5 微调 track score
-# 2023.12.05. v2.3.6 candidates 增加 cleavage site，修正 alignment 有 deletion 会错位的 bug
-# 2023.12.05. v2.3.7 用 cleavage site 代替 midpoint # 还没改完
-# 2023.12.07. v2.3.8 df_score 增加 df_exp, df_ctr 各自列。修复没 df_ctr 时的 bug。track score 用 proximal
-# 2023.12.09. v2.4.0 为了兼顾 proximal 和 overall，当 normalized overall signal 高于 2 时，增加 overall signal 的加分
-# 2023.12.09. v2.5.0 尝试新的加权位置
-# 2023.12.10. v2.6.0 加入 trackseq v4 的计算分支，即考虑 Region 内的 positive_pct，避免短而尖锐的信号
-# 2023.12.10. v2.6.1 有些非特异信号数值很大，如果在 control 组是大负数，可能导致减 control 后假高信号，因此给负数一个 clip
-# 2023.12.30. v2.7.0 增加 X_offplot 模块，用于绘图
-# 2023.12.31. v2.7.1 control 的负数值 clip 由 -5 改为 -1，进一步减少假阳性。另外不加 overall 了
-# 2024.01.01. v2.7.2 权重改为 proximal + pct = 1 + 1. 防信号外溢假阳性标准由<0改为<=0
-# 2024.01.02. v2.7.3 flank regions 默认值改为 1000 2000 3000 5000。之前 control 的负数值 clip 相当于直接在 final score，现在改为每个单独 clip 后重新算 score，默认值为 CtrClip=-0.5
-# 2024.01.03. v2.7.4 更新了 blacklist.bed
-# 2024.01.04. v2.7.5 更新了 hg38 blacklist.bed
-# 2024.01.12. v2.7.6 修复小bug，输出 fdr 改为 <0.05。
-# 2024.01.23. v2.7.7 Snakefile_offtracker: add --fixedStep to bigwigCompare for not merging neighbouring bins with equal values.
-# 2024.02.01. v2.7.8 逐步添加 X_offplot.py 功能

offtracker-2.7.8/offtracker.egg-info/PKG-INFO

@@ -1,146 +0,0 @@
-Metadata-Version: 2.1
-Name: offtracker
-Version: 2.7.8
-Summary: Tracking-seq data analysis
-Home-page: https://github.com/Lan-lab/offtracker
-Author: Runda Xu
-Author-email: runda.xu@foxmail.com
-Requires-Python: >=3.6.0
-Description-Content-Type: text/markdown
-License-File: LICENSE.txt
-
-
-OFF-TRACKER
-=======================
-
-OFF-TRACKER is an end to end pipeline of Tracking-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 requirements
------
-* Linux/Unix 
-* Python >= 3.6
-
-Dependency
------
-
-```bash
-# We recommend creating a new enviroment using mamba/conda to avoid compatibility problems
-# If you don't use mamba, just replace the code with conda 
-mamba create -n offtracker -c bioconda blast snakemake pybedtools
-```
-
-
-Installation 
------
-
-```bash
-# activate the environment
-conda activate offtracker
-
-# Direct installation with pip
-pip install offtracker
-
-# (Alternative) Download the offtracker from github
-git clone https://github.com/Lan-lab/offtracker.git 
-cd offtracker
-pip install .
-```
-
-
-Before analyzing samples
------
-
-```bash
-# Build blast index (only need once for each genome)
-makeblastdb -input_type fasta -title hg38 -dbtype nucl -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 -t 8 -g hg38 \
--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_Candidates
-
-```
-
-Strand-specific mapping of Tracking-seq data 
------
-
-```bash
-# Generate snakemake config file 
-offtracker_config.py -t 8 -g hg38 --blacklist hg38 \
--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 \ 
---subfolder 0 
-
-# --subfolder: If different samples are in seperate folders, set this to 1
-# -o: Default is outputting to /Your_Path_To_Fastq
-
-# Run the snakemake program
-cd /Your_Path_To_Fastq
-snakemake -np # dry run
-nohup snakemake --cores 16 1>snakemake.log 2>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
------
-
-```bash
-# In this part, multiple samples in the same condition can be analyzed in a single run by pattern recogonization of sample names
-
-offtracker_analysis.py -g hg38 --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)
-```
-
-
-Note1
---------------
-The 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.
-
-Note2
---------------
-Currently, 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.py
-
-Note3
---------------
-The FDR in the Tracking-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 Tracking-seq result.
-