Supporting scripts and notebooks detailing how to use ANANSE with CAGE data.

• CAGEfightR_to_ANANSE.Rmd - Rmarkdown script used to generate input files for ANANSE-CAGE from CAGE Transcription Start Site data (.ctss)

• basic_ANANSE-CAGE_run.txt - Simple example how to run ANANSE-CAGE

• ANANSE-CAGE-run.ipynb - Script used to run multiple ANANSE-CAGE analyses

• model_training_ANANSE-CAGE.ipynb - Training of binding prediction model (CAGE data only)

• model_training_ANANSE-CAGE-H3K27ac.ipynb - Training of binding prediction model (CAGE and H3K27ac data)

To perform the CAGE module of ANANSE we first need to generate the necessary input files using R, specifically the CAGEfightR package. Use this R markdown as a reference. Note, the best prediction algorithm currently only supports hg19 and hg38. We recommend to use triplicates.

First, edit the first chunk of the this R markdown according to your own data and structure. For example, make sure you use the correct assembly, working directory, paths, and names.

You can use BED files (tab seperated) with 6 columns:

1. chr
2. start
3. end
4. chr:start..end,strand
5. tag count
6. strand

For example: chr1 564586 564587 chr1:564586..564587,+ 2 +

CAGEfightR can turn this BED format and convert it to BigWig files that are then used for tag quantification. Make sure to set "PREPROCESSING = TRUE" in this R markdown script in order to convert the BED files to BigWigs.

Use the BigWig files to run the third chunk of this R markdown script. You should end up with these files:

• enhancer_source.tsv
• enhancer_target.tsv
• TPM_source_N.txt
• TPM_target_N.txt
• DE_genes.txt

Install ANANSE according to the documentation

Required layout:

• work_dir/
  • data/
    • enhancer_source.tsv
    • enhancer_target.tsv
    • TPM_source_N.txt
    • TPM_target_N.txt
    • DE_genes.txt
  • results/
    • (ouput files will be deposited here)

Perform the following steps:

ananse binding -C ./data/enhancers_source.tsv -o ./results/source_binding -g hg19;
ananse binding -C ./data/enhancers_target.tsv -o ./results/target_binding -g hg19;

ananse network ./results/source_binding/binding.h5 -e ./data/TPM_source_*.txt -o ./results/network_source.tsv -g hg19 -n 4;
ananse network ./results/target_binding/binding.h5 -e ./data/TPM_target_*.txt -o ./results/network_target.tsv -g hg19 -n 4;

ananse influence -s ./results/network_source.tsv -t ./results/network_target.tsv -d ./data/DE_genes.txt -o ./results/influence.tsv -i 500000 -n 12;

ananse plot ./results/influence.tsv -d ./results/influence_diffnetwork.tsv -o ./results/influence_results;