Measuring motif similarity is essential for identifying functionally related transcription factors (TFs) and RNA-binding proteins (RBPs), and for annotating de novo motifs. Here, we describe Motif Similarity Based on Affinity of Targets (MoSBAT), an approach for measuring the similarity of motifs by computing their affinity profiles across a large number of random sequences. We show that MoSBAT successfully associates de novo ChIP-seq motifs with their respective TFs, accurately identifies motifs that are obtained from the same TF in different in vitro assays, and quantitatively reflects the similarity of in vitro binding preferences for pairs of TFs.
- To install the program, extract the package
- Change directory:
cd MoSBAT/ - Make MoSBAT:
make - To test the pipeline, execute this command:
bash MoSBAT.sh MyTestJob \
examples/RNACompete/RNACompete.part1.pfm.txt \
examples/RNACompete/RNACompete.part2.pfm.txt rna 20 5000
This should create a “./out/MyTestJob” folder, with the MoSBAT output files described below.
To compare your motifs using MoSBAT you will need a CIS-BP formatted position frequency matrix (PFM) file. Each motif should include the following lines:
- Identifier
Motif<tab>Motif_ID - Motif Header
Pos<tab>A<tab>C<tab>G<tab>T - Position Frequency Line(s):
PositionNumber<tab>Freq_A<tab>Freq_C<tab>Freq_G<tab>Freq_T- PositionNumber starts at 1
- Each lines frequencies should sum to 1
Note: If the PFM file has more than 1 motif in it, 2 new lines should separate motifs. An example can be downloaded here, and RNA motif examples are present in examples/RNACompete.
###Usage Use the MoSBAT.sh script to run MoSBAT on your dataset:
bash MoSBAT.sh <job_name> <PFM_file_1> <PFM_file_2> <motif_type> <seq_length> <num_seqs>
Inputs:
<job_name>: Name of output folder. Results and error logs are written to out/<job_name>
<PFM_file_1> and <PFM_file_2>: Locations of CIS-BP formatted PFM files that you are comparing. Note that you can use null as <PFM_file_2>, in which case <PFM_file_1> will be compared to a built-in database of RNA/DNA motifs.
<motif_type>[rna or dna]: Whether the motifs you're comparing are RNA motifs (score in forward direction), or DNA motifs (score in forward and reverse directions)
Variable settings:
<seq_length> [recommended: 50]: To have the most accurate similarity score the length should be optimized to the: <length of motif 1> + <length of motif 2> - 1
<num_seqs> [recommended: 50000]: MoSBAT-e score variances are relatively stable for anything over 50,000 sequences. When using MoSBAT-a with long motifs we recommend >100,000 random sequences
MoSBAT outputs the following files containing all pairwise comparisons of motifs in the first set of PFMs to the second set:
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Full Matrix of MoSBAT-a Results (results.affinity.correl.txt) – Text file containing a matrix of motif similarities based on sequence affinities (MoSBAT-a). Matrix has dimensions: Motif_Set_1 by Motif_Set_2
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Full Matrix of MoSBAT-e Results (results.energy.correl.txt) – Text file containing a matrix of motif similarities based on sequence energies (MoSBAT-e). Matrix has dimensions: Motif_Set_1 by Motif_Set_2
For convenience MoSBAT also identifies the most similar pairs of motifs and their offsets. The outputs are available as heatmaps and browser viewable tables:
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Heatmap of Top MoSBAT-a Hits (results.affinity.correl.heatmap.jpg) – Heatmap image displaying at most top 10x10 MoSBAT-a (results.affinity.correl.txt) motif similarity values.
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Table of Top MoSBAT-a Hits (results.affinity.correl.htm) – HTML table of the top 1000 pairs of motifs (results.affinity.correl.txt). Includes MoSBAT-a scores, and a histogram of offsets for the top 100 pairs of motifs.
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Heatmap of Top MoSBAT-e Hits (results.energy.correl.heatmap.jpg) – Heatmap image displaying at most top 10x10 MoSBAT-e (results.energy.correl.txt) motif similarity values.
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Table of Top MoSBAT-e Hits (results.energy.correl.htm) – HTML table of the top 1000 pairs of motifs (results.energy.correl.txt). Includes MoSBAT-e scores, and a histogram of offsets for the top 100 pairs of motifs.
Changes to the random sequences that MoSBAT uses can be done by editing the seq_template value on line 7 of the MoSBAT.sh file. To do this, change the location from the default random sequence file to your FASTA file of interest. Any run of MoSBAT using that bash file will calculate motif similarity using the fist L bases specified by the <seq_length> parameter from each of the first N sequences specified by the <num_seqs> parameter.
MoSBAT does not calculate the significance of motif similarities but it can be generated using the outputs from the tool. First a large collection of random motifs needs to be created as a background. One way is to generate a large random set of motifs with similar characteristics to your query using the method of Sandelin and Wasserman (2004). The -genrand function to make your random set of motifs implemented in the STAMP toolkit by Mahony, Auron, and Benos (2007) can be used to do this.
Next, run MoSBAT using the background motif collection, and use the generated set of MoSBAT-a and MoSBAT-e scores as the background (null) distribution to calculate the p-values for other motif pairs.
Lambert SA, Albu M, Hughes TR, Najafabadi HS. Motif comparison based on similarity of binding affinity profiles. Bioinformatics 2016, doi:10.1093/bioinformatics/btw489