ATAC-seq (Assay for Transposase-Accessible Chromatin using high-throughput sequencing) is a sequencing assay for investigating genome-wide chromatin accessibility. The assay applies a Tn5 Transposase to insert sequencing adapters into accessible chromatin, enabling mapping of regulatory regions across the genome. Additionally, the local distribution of Tn5 insertions contains information about transcription factor binding due to the visible depletion of insertions around sites bound by protein - known as footprints.
TOBIAS is a collection of command-line bioinformatics tools for performing footprinting analysis on ATAC-seq data, and includes:
- Correction of Tn5 insertion bias
- Calculation of footprint scores within regulatory regions
- Estimation of bound/unbound transcription factor binding sites
- Visualization of footprints within and across different conditions
For information on each tool, please see the wiki.
TOBIAS is written as a python package and can be quickly installed within a conda environment using:
$ git clone https://github.molgen.mpg.de/loosolab/TOBIAS
$ cd TOBIAS
$ conda env create -f snakemake_pipeline/environments/tobias.yaml
$ conda activate TOBIAS_ENV
$ python setup.py installPlease see the installation page for more info.
All tools are available through the command-line as TOBIAS <TOOLNAME>, for example:
$ TOBIAS ATACorrect
__________________________________________________________________________________________
TOBIAS ~ ATACorrect
__________________________________________________________________________________________
ATACorrect corrects the cutsite-signal from ATAC-seq with regard to the underlying
sequence preference of Tn5 transposase.
Usage:
TOBIAS ATACorrect --bam <reads.bam> --genome <genome.fa> --peaks <peaks.bed>
Output files:
- <outdir>/<prefix>_uncorrected.bw
- <outdir>/<prefix>_bias.bw
- <outdir>/<prefix>_expected.bw
- <outdir>/<prefix>_corrected.bw
- <outdir>/<prefix>_atacorrect.pdf
(...)
These examples use the test data provided in the TOBIAS/test_data directory, so please make sure you are in the upper TOBIAS/ directory when running the commands.
ATACorrect: Bias correction of ATAC-seq reads in open chromatin
$ TOBIAS ATACorrect --bam test_data/Bcell_chr4.bam --genome test_data/genome_chr4.fa.gz --peaks test_data/merged_peaks.bed --blacklist test_data/blacklist_chr4.bed --outdir atacorrect_test --prefix Bcell --cores 8
FootprintScores: Calculate footprint scores from corrected cutsites
$ TOBIAS FootprintScores --signal test_data/Bcell_corrected.bw --regions test_data/merged_peaks.bed --output Bcell_footprints.bw --cores 8
BINDetect: Estimation of differentially bound motifs based on scores, sequence and motifs
$ TOBIAS BINDetect --motifs test_data/example_motifs.txt --signals test_data/Bcell_footprints.bw test_data/Tcell_footprints.bw --genome test_data/genome_chr4.fa.gz --peaks test_data/annotated_peaks.bed --peak_header test_data/annotated_peaks_header.txt --outdir bindetect_output --cond_names Bcell Tcell --cores 8
PlotAggregate: Plot aggregated ATAC-seq signals in combinations of .bed/.bw to visualize footprints
Visualize the difference in footprints between two conditions for all accessible sites:
$ TOBIAS PlotAggregate --TFBS test_data/BATFJUN_all.bed --signals test_data/Bcell_corrected.bw test_data/Tcell_corrected.bw --output BATFJUN_footprint_comparison_all.pdf --share_y both --plot_boundaries
Visualize the difference in footprints between two conditions exclusively for bound sites:
$ TOBIAS PlotAggregate --TFBS test_data/BATFJUN_Bcell_bound.bed test_data/BATFJUN_Tcell_bound.bed --signals test_data/Bcell_corrected.bw test_data/Tcell_corrected.bw --output BATFJUN_footprint_comparison_subsets.pdf --share_y both --plot_boundaries
Visualize the split of bound/unbound sites for one condition:
$ TOBIAS PlotAggregate --TFBS test_data/IRF1_all.bed test_data/IRF1_bound.bed test_data/IRF1_unbound.bed --signals test_data/Bcell_uncorrected.bw test_data/Bcell_expected.bw test_data/Bcell_corrected.bw --output IRF1_footprint.pdf --share_y sites --plot_boundaries
PlotHeatmap: Plot heatmaps and aggregates of ATAC-seq signals in combinations of .bed/.bw to visualize footprints
$ TOBIAS PlotHeatmap --TFBS test_data/BATFJUN_Bcell_bound.bed test_data/BATFJUN_Bcell_unbound.bed --TFBS test_data/BATFJUN_Tcell_bound.bed test_data/BATFJUN_Tcell_unbound.bed --signals test_data/Bcell_corrected.bw test_data/Tcell_corrected.bw --output BATFJUN_heatmap.pdf --signal_labels Bcell Tcell --share_colorbar
FormatMotifs: A utility to convert and join/split across different motif-file formats
Join individual motif files to one:
$ TOBIAS FormatMotifs --input test_data/individual_motifs/* --format pfm --task join --output example_motifs.txt
Split a motif file containing several motifs:
$ TOBIAS FormatMotifs --input test_data/example_motifs.txt --format pfm --task split --output split_motifs
You can run each TOBIAS tool independently or as part of a pipeline using the included snakemake workflow. To use the snakemake pipeline, make sure the included conda environments are installed:
$ conda env create -f snakemake_pipeline/environments/tobias.yaml
$ conda env create -f snakemake_pipeline/environments/macs.yaml
You can use the example config (snakemake_pipeline/TOBIAS_example.config) or adjust to your own data by replacing the values for each key. Run using:
$ cd snakemake_pipeline
$ conda activate TOBIAS_ENV
$ snakemake --snakefile TOBIAS.snake --configfile TOBIAS_example.config --cores [number of cores] --keep-goingFor further info on setup, configfile and output, please consult the wiki.
This project is licensed under the MIT license.
Mette Bentsen (mette.bentsen (at) mpi-bn.mpg.de)