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FAQ
The TOBIAS tools are intended to be run in the order of:
- ATACorrect
- ScoreBigwig
- BINDetect
You can also check out the pre-set snakemake pipeline in order to automate analysis for many conditions.
You can use any .bam-file containing ATAC-seq reads. It is recommended that you remove PCR duplicates, as these can otherwise influence footprinting. You, however, do not need to shift the reads +4/-5, as TOBIAS ATACorrect does this internally.
You should use any .bed-file containing open chromatin regions from peak-calling, e.g. from MACS2 or similar. If you are planning to compare several conditions with each other, e.g. 'WT.bam' with 'treatment.bam', you should obtain the peaks 'WT_peaks.bed' and 'treatment_peaks.bed' for each condition, and merge these using e.g. bedtools:
cat WT_peaks.bed treatment_peaks.bed | bedtools sort | bedtools merge > merged_peaks.bed
You should then use 'merged_peaks.bed' throughout the TOBIAS tools.
TOBIAS does not deal with individual biological replicates, and it is therefore recommended to merge replicate .bam-files prior to correction and footprinting. This is to improve the sequencing depth, as well as simplify the downstream interpretation of the results.
TOBIAS BINDetect and other tools require files per motif/TF to be open throughout the run, and this can exceed the system-limit for open file handles. This can be fixed using the command ulimit:
$ ulimit -n 3000 (or the amount needed - this scales with the number of motifs used)
Please search the issues page to see if someone has already asked a similar question. Otherwise, please feel free to open a new issue.