ATAC-seq

Summary

The ATAC-seq pipeline first trims the raw reads to remove Tn5 adaptor sequence using skewer. Then reads are mapped to the genome (-g) using BWA mem. Raw mapped reads are labeled as .markdup.bam. De-duplicated reads are labeled as .rmdup.bam. De-duplicated and uniquely mapped reads are labeled as .rmdup.uq.bam. Duplicated and multi-mapped reads were removed using samtools (v0.17). ATAC-seq peaks were called using MACS2 (v2.1.1) with the following parameters “macs2 callpeak –nomodel –shift -100 –extsize 200”. BigWiggle files were generated using DeepTools bamCoverage (v3.2.0) with “–centerReads”.

Code for this pipeline is provided in “https://github.com/YichaoOU/HemTools/blob/master/subcmd/”. See atac_seq.py and utils.py.

Parameters

usage: HemTools atac_seq [-h] [-j JID] [--short] [--debug]
                         (-f INPUT | --guess_input) [-i INDEX_FILE]
                         [-g GENOME] [-b BLACKLIST] [-s CHROM_SIZE]
                         [-e EFFECTIVEGENOMESIZE]

Named Arguments

-j, --jid

enter a job ID, which is used to make a new directory. Every output will be moved into this folder.

Default: “{{subcmd}}_docs_2024-03-15”

--short

Force to use the short queue. (only if R1+R2 fastq.gz size <=250M)

Default: False

--debug

Not for end-user.

Default: False

-f, --input

tab delimited 3 columns (tsv file): Read 1 fastq, Read 2 fastq, sample ID

--guess_input

Let the program generate the input files for you.

Default: False

Genome Info

-i, --index_file

BWA index file

Default: “/home/docs/checkouts/readthedocs.org/user_builds/hemtools/checkouts/latest/subcmd/../hg19/bwa_16a_index/hg19.fa”

-g, --genome

genome version: hg19, hg38, mm10, mm9.

Default: “hg19”

-b, --Blacklist

Blacklist file

Default: “/home/docs/checkouts/readthedocs.org/user_builds/hemtools/checkouts/latest/subcmd/../hg19/Hg19_Blacklist.bed”

-s, --chrom_size

chrome size

Default: “/home/docs/checkouts/readthedocs.org/user_builds/hemtools/checkouts/latest/subcmd/../hg19/hg19.chrom.sizes”

-e, --effectiveGenomeSize

effectiveGenomeSize for bamCoverage

Default: “2451960000”

Flowchart

../../_images/atac_seq_pipeline.png

Usage

Go to your data directory and type the following.

Step 0: Load python version 2.7.13.

$ module load python/2.7.13

Step 1: Prepare input files, generate fastq.tsv.

$ HemTools atac_seq --guess_input

    Input fastq files preparation complete! ALL GOOD!
    Please check if you like the computer-generated labels in : fastq.tsv

Note

If you are preparing fastq.tsv yourself, please make sure no space anywhere in the file. Note that the seperator is tab. Spaces in file name will cause errors.

Step 2: Check the computer-generated input list (manually), make sure they are correct.

$ less fastq.tsv

Note

a random string will be added to the generated files (e.g., fastq.94c049cbff1f.tsv) if they exist before running step 1.

Step 3: Submit your job.

$ HemTools atac_seq -f fastq.tsv

Sample input format

fastq.tsv

This is a tab-seperated-value format file. The 3 columns are: Read 1, Read 2, sample ID.

../../_images/fastq.tsv.png

Quality Control

The quality metrics are provided in the html report. For ChIP-seq data, we also provide strand cross-correlation metrics (i.e., those attached pdf files).

Metrics

Threshold

NRF

>0.9

PBC1

>0.9

PBC2

>3

Num peaks

>100k

https://www.encodeproject.org/atac-seq/

https://www.encodeproject.org/chip-seq/transcription_factor/ https://www.encodeproject.org/chip-seq/histone/

https://github.com/crazyhottommy/ChIP-seq-analysis/blob/master/part0_quality_control.md

Report bug

Once the job is finished, you will be notified by email with some attachments. If no attachment can be found, it might be caused by an error. In such case, please go to the result directory (where the log_files folder is located) and type:

$ HemTools report_bug

Use different genome index

$ HemTools atac_seq -f fastq.tsv -i YOUR_GENOME_INDEX

Example of using different genome index

Ruopeng masked index

../../_images/ruopeng_mask.PNG
HemTools atac_seq -f fastq.tsv -i /home/yli11/Data/Human/hg19/index/masked_genome/ruopeng_hbg1_promoter/ruopeng_hbg1_promoter.mask.fa

Li masked index

../../_images/Li_mask.PNG
HemTools atac_seq -f fastq.tsv -i /home/yli11/Data/Human/hg19/index/masked_genome/li_hgb1_promoter/li_hgb1_promoter.mask.fa

Comments

code @ github.