Cut and Tag Pipeline

Background

This Cut and Tag Pipline based on

CUT&Tag Data Processing and Analysis Tutorial from Ye Zheng el. _al.

Prerequisites

conda is used to mange software used in this pipeline. For further information, please consult https://docs.anaconda.com/anaconda/install/linux-aarch64/.

Included in this repository is the conda_env_no_build.yaml which contains all packages required to install this pipeline.

Installation

1. Clone the repo

   git clone 

2. Activate conda environment with RNA_Seq.yml file

   conda env activate -f conda_env_no_build.yaml

You are now ready to run the pipeline!

Nextflow

Under development

Bash

Example bash script can be found in example folder

Core

use script/main/run_cut_tag.sh to run cut and tag pipeline.

Input

The script takes the following arguments:

• raw_fastq1
• raw_fastq2
• out_dir
• sample_name
• script_dir
• skip_trimmed
• no_rose2
• no_spikein
• peak_caller ( seacr or macs2 )
• ctrl_bedgraph
• ctrl_bam

bash script/main/run_cut_tag.sh $fastq_1 $fastq_2 ${out_dir}/$sample_name $sample_name $script_dir false true $ctrl_bam $ctrl_bedgraph 

Post-processing

Run QC

bash script/post_processing/run_QC.sh script/post_processing/QC $out_dir

Organize all outputs

Input

• out_dir: directory where the pipeline output directory is
• final_result: path where organized data folder should be

bash $script_folder/symlink_final_result.sh $out_dir $final_result

Output

Single sample output

Sample_folder
├── alignment
│   ├── bam
│   └── bed
├── fastqc
├── peak_calling
├── QC_summary
└──trimmed_reads

Organized data output

final_results
├── bam
├── cpm
├── fastqc
├── frag_legnth
├── Sample_run_frip_score_summary.txt
├── Sample_run_QC_summary.csv
├── peak
└── QC

Note: all reference files should be downloaded to ref folder

• NEB Ultra II Kit is obtained from https://www.neb.com/faqs/2021/01/15/what-sequences-need-to-be-trimmed-for-nebnext-libraries-that-are-sequenced-on-an-illumina-instrument (If you use other library prep kit other than NEB, you need to obtain adapter sequence from the vendor)
• Bowtie2 genome ref should be downloaded into ref folder. We use the no alternative set. https://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#indexing-a-reference-genome
• E. coli genome ref should be downloaded into ref folder if you use E. coli spike-in

Pooled Peak calling

Inspired by ChIP-seq ENCODE 3 pipeline, replicated BAM files are combined for peak calling using MACS2. IDR is then applied to assess the reproducibility of the pooled peak sets.

Run MACS2

The script takes the following arguments:

• out_sample : output sample name with path (e.g. out_folder/Sample1_pooled)
• control_bam
• sample_repX_bam : bam files for reach replicates

bash script/pooled_sample_processing/pooled_run_macs2.sh $out_sample $control_bam $sample_rep1_bam $sample_repX_bam 

Run IDR

The script accept only two replicates peak sets and run by using the following argument.

• rep1
• rep2
• pool_peak
• Idr_output_folder

bash script/pooled_sample_processing/run_idr.sh $rep1 $rep2 $pooled_peak $idr_output