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Annotating regions of the genome for occurences of DNA motifs (e.g transcription factor DNA binding sites)
Input:
- Regions of the genome to annotate, typically promoter regions (.bed)
- Columns: `chrom`, `window_start`, `window_end`, `identifier`
where the identifier is typically the ENSEMBL gene ID
- Make sure that window_start and window_end are never out of the 0:chrom_size interval, otherwise twoBitToFa will raise an error. Note that UCSC tools all use 0-based indexing, with the end coordinate *not* included in the sequence.
- Reference genome
- MEME-formatted list of position-weight matrices (.txt)
- Number of cores for multiprocessing
- p-value threshold for FIMO
Output:
- Concatenated .txt file of FIMO output results, for further processing in pandas or R.
Key steps:
- Split the .bed file into multiple .bed files according to the number of cores used for multiprocessing
- bedtools2/tools/split, equal records matters.
- Extract the fasta sequences from the reference genome, using the bedfile as an input:
- twoBitToFa, with the .bed regions and the .2bit genome as inputs.
- Compute the background frequencies for the regions of interest and store the results in a 0-order Markov background model as specified by the MEME suite
- Launch parallel processes of FIMO on the newly created .fasta files.
- FIMO is case insensitive -> no risks with masked regions
- Concatenate FIMO outputs into a single .txt file
- Sort FIMO outputs by gene, motif, chr and start.
- Save results on /home
Practical notes:
- Use environment variables: $HOME, $USER for compatibility with other usernames
- Work in /scratch/$USER/, save results in $HOME
- Running the annotation on 57700 human genes with 5500+ motifs ran in a bit more than 70 minutes
Reproduce the work on SCITAS:
0) check the SCITAS modules and python 3 requirements needed.
- Load modules and activate source environment
1) execute 'download_ref_genome.py'
2) execute 'download_ucsc_binaries.py'
3) execute 'split_bed_files.py'
4) execute 'extract_fasta.py'
5) modify and execute 'run_fimo.sh'
- launches run_fimo.py
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