Incorporating CL-motif scores

In order to address the crosslinking sequence bias, PureCLIP can incorporate information about motifs that are known to be preferentially crosslinked, also called CL-motifs (see also Haberman et al., 2017). For each CL-motif PureCLIP learns the influence on the crosslinking probability. This can be particular useful for proteins binding to sequence motifs distinct from such CL-motifs.

Compute CL-motif scores

To incorporate CL-motifs into the model of PureCLIP, first we need to compute position-wise CL-motif scores, indicating the positions CL-affinity. You can use the provided precompiled list of common_CL-motifs:

wget -O motifs.txt https://raw.githubusercontent.com/skrakau/PureCLIP_data/master/common_CL-motifs/dreme.w10.k4.txt
wget -O motifs.xml https://raw.githubusercontent.com/skrakau/PureCLIP_data/master/common_CL-motifs/dreme.w10.k4.xml

If you want to compute the CL-motifs specific to the used eCLIP experiment, please have a look here. Assume we have given a set of CL-motifs, we use FIMO (Grant et. al, 2011) to compute motif occurrences associated with a score within the reference sequence. The following provided script (distributed with PureCLIP) first retrieves reference regions covered by the target experiment, then runs FIMO to compute position-wise CL-motif match scores within such regions and chooses for each position the motif with the highest score:

export BEDTOOLS=/path/to/bedtools        # if not specified, PATH is searched
export FIMO=/path/to/fimo                # if not specified, PATH is searched
export WINEXTRACT=/path/to/winextract    # built together with PureCLIP
compute_CLmotif_scores.sh ref.hg19.fa aligned.prepro.R2.bam motifs.xml motifs.txt fimo_clmotif_occurences.bed

The resulting CL-motif scores are written to fimo_clmotif_occurences.bed.

PureCLIP

The computed scores are then handed over to PureCLIP together with the parameter -nim 4, indicating that scores with associated motif IDs 1-4 will be used (default: only scores with motif ID 1 are used).

pureclip -i aligned.prepro.R2.bam -bai aligned.prepro.R2.bam.bai -g ref.hg19.fa -o PureCLIP.crosslink_sites.cov_CLmotifs.bed -nt 10 -iv 'chr1;chr2;chr3;' -nim 4 -fis fimo_clmotif_occurences.bed