Approximate time: 20 minutes

Preparing files for Motif Analysis

Once you have your set of enriched regions which represent binding sites from your protein of interest, a next logical question is within those regions are there any similar patterns of sequences that are over-represented. These sequences are often referred to as motifs.

Tools for motif analyis often require sequence information for each of your binding regions, and so this lesson will show you how to use bedtools to obtain that.

Start an interactive session:

$ srun --pty -p interactive -t 0-12:00 --mem 1G --reservation=HBC2 /bin/bash

Since the motif analyses are unlikely to give reliable results using only the 32.8 Mb of reads mapping to chr12, we will use the full set of peak calls output from the IDR analysis. Move into your project directory, and copy over the IDR bed files:

$ cd ~/chipseq/results

$ cp /n/groups/hbctraining/chip-seq/full-dataset/idr/*.bed .

Extract the first three columns of the IDR peak calls for the Nanog file:

	$ cut -f 1,2,3 Nanog-idr-merged.bed  > Nanog-idr-merged-simple.bed

To extract the sequences corresponding to the peak coordinates for motif discovery, we will use the bedtools suite of tools. The getfasta command extracts sequences from a reference fasta file for each of the coordinates defined in a BED/GFF/VCF file.

$ module load gcc/6.2.0 bedtools/2.27.1	

$ bedtools getfasta -fi \
/n/groups/shared_databases/igenome/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa \
-bed Nanog-idr-merged-simple.bed \
-fo Nanog-idr-merged-dreme.fasta

Using scp or FileZilla on your local computer, transfer Nanog-idr-merged-dreme.fasta to your Desktop.

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This lesson has been developed by members of the teaching team at the Harvard Chan Bioinformatics Core (HBC). These are open access materials distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.