Many of the hotspot mutations were located in the 5′ flanks of the consensus CTCF motif (Fig. 4f). Previous studies have found increased conservation in the flanking sequences of weak CTCF and REST binding sites, suggesting that the sequence context is important for TF binding at these sites52,53. We examined the evolutionary conservation54 at the CTCF binding motifs and their flanking sequences. In general, the 5′ flanks of the CTCF motifs are not conserved (Supplementary Fig. 13a). However, in the hotspot upstream of CENPQ, the mutation cluster in the 5′ flank co-occurred with conserved bases (Supplementary Fig. 13b). In addition, we found another CBS hotspot with nine 5′-flank mutations that coincided with a highly conserved base (Supplementary Fig. 13c). Such hotspot mutations, affecting conserved 5′ flanks of CTCF motifs, could disrupt context-specific binding of CTCF.