FAQ

FAQ (Frequently Asked Questions)

1. What is the recommended workflow for the TOBIAS tools? The TOBIAS tools are intended to be run in the order of:

• ATACorrect
• ScoreBigwig
• BINDetect Check out the pre-set snakemake pipeline in order to automate analysis for many conditions: https://github.molgen.mpg.de/loosolab/TOBIAS_snakemake

2. What .bam-file should I use as input? 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.

3. What peak-file should I use as input? 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.bam treatment.bam | bedtools sort | bedtools merge > merged_peaks.bed You should then use merged_peaks.bed throughout the TOBIAS tools.

4. How do I deal with replicates? 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.

5. I get an error stating "too many files open" - what do I do? 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)

6. I have another question not answered here... Please search the https://github.com/loosolab/TOBIAS/issues to see if someone has already asked a similar question. Otherwise, please feel free to write a new issue on the page.