Tn5DP (Tn5-based epigenetic Data Processing)

Pipeline for the QC metrics construction, data analysis and visualization of ATAC-seq and CUT&Tag-seq data.

Current Version: Tn5DP_v1.0 Last update: 2022.10.12

Advisor: Bo Zhang
Contributor: Siyuan Cheng

For any question, please contact siyuancheng@wustl.edu 👈

Documentation

1. Pipeline documentation: analysis details, parameters, and QC metrics information
   Please click here
2. Update logfile: pipeline change record
   Please click here
3. Check the definition of narrow/broad marker, and mapped-read numbers
   Please click here

Usage:

Singularity3 Installation

Tn5DP image has to be run with Singularity version3+, you could follow this instruction if you haven`t install Singularity3.
Please click here
(You will need sudo permission to properlly install and configure it, but you can run it without sudo after installation:smiley:)

Test ATAC-seq data:

There is one paired-end mm10 data with 0.25M reads for test purpose, they can be downloaded by:

wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/sample_data/atac-seq/mm10_1.fastq.gz
wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/sample_data/atac-seq/mm10_2.fastq.gz

Test CUT&Tag-seq data:

There are one paired-end mm10 data (H3K36me3 marker) with 0.25M reads for test purpose, they can be downloaded by:

wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/sample_data/cuttag-seq/mm10_k36me3_1.fastq.gz
wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/sample_data/cuttag-seq/mm10_k36me3_2.fastq.gz
wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/sample_data/cuttag-seq/mm10_igg_1.fastq.gz
wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/sample_data/cuttag-seq/mm10_igg_2.fastq.gz

Run Tn5DP:

Step1 Download the singularity image and reference files (you only need download them ONCE, then you can use them directly), if there is any update, you may need to download a new image, but reference files are usually NOT changed:

1. Download the singularity image:

wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/Tn5DP_v1.0.simg

2. Download the reference files of different genome:

wget http://regmedsrv1.wustl.edu/Public_SPACE/ryan/Public_html/singularity_ac/Genome/mm10.tar.gz

You can also find more genome builds: click here . Currently we have: mm9/10/39, hg19/38, danRer10/11, rn6 and dm6.

3. Decompress the reference files and put to your own folder:

tar -zxvf mm10.tar.gz

Step2 Process data by the singularity image:

‼️Please run the cmd on the same directory of your data, if your data is on /home/example, then you may need cd /home/example first. The location of image and reference files is up to you.

singularity run -B ./:/home -B <path-to-parent-folder-of-ref-file>:/cuttag_atac/Resource/Genome <path-to-downloaded-image> \
-d <ATAC/CUTnTag> -g <hg38/mm10 etc.> -r <PE/SE> -m <narrow/broad> \
-o <experimental read file1>  -O <experimental read file2> \
-i  <IgG_control read file1>  -I <IgG_control read file2> \
-s  <AIAP/MACS2/F-seq/SEACR/HMMRATAC/ChromHMM> \
--personalize (use this option when using customized peakcalling parameter setting, default is False) \
--peakcalling options (if you use --personalize option)

For example, if
a) you download the image on /home/image/Tn5DP_v1.0.simg
b) the reference file on /home/src/mm10
c) and your data type is ATAC-seq data
d) and experiment data is read1.fastq.gz and read2.fastq.gz on folder /home/data
e) and input data is igg_1.fastq.gz and igg_2.fastq.gz on folder /home/data
f) and you want to run the recommended pipeline for ATAC-seq data

Then you need to:

1. cd /home/data
2. singularity run -B ./:/home -B /home/src:/cuttag_atac/Resource/Genome /home/image/Tn5DP_v1.0.simg -d ATAC -g mm10 -r PE -o read1.fastq.gz -O read2.fastq.gz -i igg_1.fastq.gz -I igg_2.fastq.gz -s AIAP

Note:
for ATAC-seq data, we recommend AIAP for peak calling,
for CUT&Tag-seq narrow marker data, we recommend MACS2 for peak calling,
for CUT&Tag-seq broad marker data, we recommend ChromHMM for peak calling
👉for the details of personalized peak calling options, you can check here

Step3 Differential analysis (if needed)

We use edgeR and peakcalling results, as well as BED files generated by Tn5BP to do DBR (Differeital Binding Regions) analysis. We support the user-defined value for CPM, q-value, and log2FC parameters

# similar to DEG analysis, you will need 2 conditions to compare, and sample size for each condition is at least 2, unbalanced design is supported.
# please put read file (step3.3*open.bed in ATAC-seq and step3.1*.extended.bed in CUT&Tag-seq) and peak file (*step4.1_peakcall_*) of the compared data into same folder
# please make sure for each condition, there is a keyword to group them together, namely, use ls *keyword1* can catch all read and peak files in group1, and ls *keyword2* can catch all read and peak files in group2

# run DBR analysis
singularity exec <path-2-singularity-image> bash /cuttag_atac/Resource/pipe_code/DBR_analysis.sh keyword1 keyword2 cpm_value q_value log2FC_value

For example, after running Tn5DP:
a) create a folder for DBR analysis mkdir /home/dbr_analysis
b) copy all step4.1* files to /home/dbr_analysis
c) Prepare read bed files (step3.3open.bed in ATAC-seq and step3.1.extended.bed in CUT&Tag-seq)
d) find the keywords for 2 conditions. Let's assume the keywords for two groups will be d0 and d2
e) choose CPM_value, q-value, and Log2FC_value. Let's assume you deem them as 1, 0.05, 1.5

Then you need to:

1. cd /home/dbr_analysis
2. singularity exec /path-to-image/Tn5DP_v1.0.simg bash /cuttag_atac/Resource/pipe_code/DBR_analysis.sh d0 d2 1 0.05 1.5