Motif Scan and Enrichment Analysis (MoSEA)

MoSEA Modules:

(Please refer test_files/run_test_files.sh to run the pipeline and to run MoSEA directly with SUPPA events).

I. getfasta: Extract fasta sequences from given bedfile coordinates.

Required Input: 
		 --bedfile : Bed file in atleast 6 columns format, Default:strandness is TRUE
		 --genome : Genome Fasta File sequence in single line, eg. hg19.fa
Optional Input:
		 --output : Output file name, Default: STDOUT
		 --bedtoolspath : Bedtools bin path, Default: search for 'bedtools' in local path

Output: 1. Sequence Fasta file

Usage: 
python mosea.py getfasta --bedfile sample.bed --genome /path/to/hg19.fa --output sample_output.fa
Usage (with optional parameter):
python mosea.py getfasta --bedfile sample.bed --genome /path/to/hg19.fa --output sample_output.fa --bedtoolspath /path/to/bedtools/bin/bedtools

II. scan: Scans fasta Sequences for given motifs (PFMs)

Required Input: 
		 --scan : Scans for given motif (this option is always true)	

                     --pfm : If selected; Scans for motif using PFMs
                     --pfm_path : Path to folder where PFM matrices of motifs are stored 
                    
                     --kmer : If selected; Scans for motif using k-mers 
                     --kmer_path: Path to k-mer(s) folder, (Note: kmers file should be saved with *.kmer extension, in fasta format or just one kmer in each line. Motif-label will be taken from file_name before ext (eg. for file 'SRSF1.kmer', label will be 'SRSF1').

		 --fasta : Input Fasta file (Sequences in Fasta file format to scan motifs)
		 
		
Optional Input:
		 --count : Also creates count_file in tabular format for all motifs found on each sequences.
		 --out_dir : Output Directory (path to output directory, Default: creates fmo/ directory in current working path)
		 --fmo_path : Path to FIMO binary folder, Default: Search for 'fimo' in local path

Output: 1. fmo output files for each motif in out_dir/
		2. Summary count_file table for all motifs and all sequences in one file (if --count)}

Usage: 
python mosea.py scan --pfm --pfm_path /path/to/motif_pfm_dir/ --fasta sample_output.fa
or
python mosea.py scan --kmer --kmer_path /path/to/motif_kmer_dir/ --fasta sample_output.fa 

Usage (with optional parameter):
python mosea.py scan --scan --pfm --pfm_path /path/to/motif_pfm_dir/  --fasta sample_output.fa  --out_dir /path/my/fmo_output_dir/ --fmo_path /usr/local/bin/fimo --count

III. enrich : Motif enrichment test analysis between given regulated and background(control) files

Required Input: 
		 --reg_fa_file : Input file for regulated sequences in fasta format
		 --reg_count_file : Input file for regulated motif counts in tab seperated format(header=Motif_ids)
		 --bg_fa_file : Input file for background (control) sequences in fasta format
		 --bg_count_file : Input file for background (control) motif counts in tab seperated format(header=Motif_ids)
		
Optional Input:
		 --out_file : Creates output for enrichment test analysis, Default: output_MoSEA_enrichment.txt
		 --rand_count : Number of times randomization performed for each regulated sequence. Default:100
		 --match_len : Match length of background sequences to each regulated sequence(1=True, 0=False). Default: 0 (False)
		 --len_ext : Percent range of length extension on both side boundaries, to match with background sequences.
		 			 This option is valid only when '--match_len 1'. 
					 Default: 15 (15% length extended on both sides)

Output: 1. Enrichment test output file for each motif with zscore 

Usage: 
python mosea.py enrich --reg_fa_file reg_sample_output.fa --reg_count_file reg_sample_output.space
					   --bg_fa_file bg_sample_output.fa --bg_count_file bg_sample_output.space
					 
Usage (with optional parameter):
python mosea.py enrich --reg_fa_file reg_sample_output.fa --reg_count_file reg_sample_output.space
					   --bg_fa_file bg_sample_output.fa --bg_count_file bg_sample_output.space
					   --out_file my_output_file.tab --rand_count 200 --match_len 1 --len_ext 20

Run MoSEA on Suppa events to get enriched Motifs

Requirements:

- 	Python 2.7 (numpy, bio, pandas, StringIO, collections, math)
- 	Bedtools
- 	Fimo (MEME-Suite version >version 4.11 or higher)
- 	Genome fasta sequence (\*.fa and \*.fai files)
- 	Motif PFMs or Kmers file or directory 

Input files:

- 	Suppa event ids for regulated set (or Bedfile cordinates)
- 	Suppa event ids for control set (or Bedfile cordinates)

The analysis is divided into two steps, A) Motif Scan B) Enrichment

For Analysis (A): Motif Scan Steps:

---

Input: 2 files:

• Regulated & Control event ids (Suppa events or Bedfile format)

Steps: Input_file -> Extract Sequence -> Scan sequences for Motif -> Create count table Requirement: Genome Fasta file (hg19.fa), Bedtools, FIMO from MEME suite

For Analysis (B): Enrichment

---

Input: 4 files: (All these files are generated in Analysis A)

• Regulated fasta sequences file
• Regulated motif count table
• Control(Background) fasta sequence file
• Control motif count file

Steps: For each regulated sequence -> create pools of Control seqs matching GC content and length of seq -> randomize 100 times -> count motifs on reg & control -> calculate z-score by observe(reg) vs expected (distribution from control) ((x-mean)/SD)

To test run call this script from MoSEA directory:

cd MoSEA/
./test_files/run_test_files.sh (Please point the paths to appropriate directories)

---

Analysis (A): Motif Scan

###Commands:

Step1 : Convert Suppa events to bedfile format

(This step is not necessary if cordinates are already in bedfile format. Go to step 2 directly)

$path_python ./mosealib/suppa_to_bed.py --ifile $reg_infile --event SE --ext 200 --ofile $bedfile_reg

(Ext : Extension is upstream and downstream regions from the center Exon)

Step2: Convert Bedfile to Fasta File

$path_python mosea.py getfasta --bedfile $bedfile_reg --genome $path_genome --output $fafile_reg

Step3: Scan fasta sequences for Motifs (Example shown for PFMs)

fmopfm_outdir="fmo_pfm"  #output dir for scanned motifs
$path_python mosea.py scan --pfm --pfm_path $path_pfms --fasta $fafile_reg --out_dir $fmopfm_outdir --fmo_path $path_fimo --count

Repeat above three steps for control files as well. Ideally, number of events in control set must be atleast 100x more than regulated set.

Analysis (B): Motif Enrichment Analysis

Required Input: 4 files (2 regulated files: fasta & count_table, 2 control files: fasta & count_table) generated in Analysis (A).

Important: Only in case of running SUPPA events, please seperate variable region types prior to each enrichment analysis. For eg.

1. To seperate upstream region of SE event from fasta file (regulated and control):

grep -A1 ';up' regfile.fa >events_SE_up_reg.fa
grep -A1 ';up' controlfile.fa >events_SE_up_control.fa

2. To seperate upstream region of SE event from count file (regulated and control):

cat reg_file_count | head -1 >up_reg_file_count; grep ';up' reg_file_count >>up_reg_file_count
cat control_file_count | head -1 >up_cont_file_count; grep ";up" control_file_count >>up_cont_file_count

Repeat it for all regions. (Please see run script test_files/run_script_test_all_events.sh for more information)

Step4: Perform Enrichment

Regulated files : Fasta sequences and Motif count table (per region type)
- reg_file_fa
- reg_file_count
	
Control files : Fasta sequences and Motif count table (per region type)
- control_file_fa
- control_file_count

#Zscore Output file name
outfile="zscore_outfile.tab"

#perform enrichment
$path_python mosea.py enrich --reg_fa_file $reg_file_fa --reg_count_file $reg_file_count \
                       --bg_fa_file $control_file_fa --bg_count_file $control_file_count \
		     	--out_file $outfile

---

To run the pipeline all at once, please run the dummy script in test_files/run_test_files.sh To run extended version of all SUPPA events with variable region, please run script: test_files/run_script_test_all_events.sh