Focusing on the TF-bound instances of Twi, Bin and Tin motifs, we first analyzed sequence variation at each motif position across 12 Drosophila species (Figure 1a) and across D. melanogaster individuals (Figure 1b). As expected, TF-bound motifs both are conserved across evolutionary distance and show depressed levels of variation across individuals compared to either their respective flanking regions (Figure 1a,b), reshuffled motifs, unbound motifs or the third bases of Gly codons considered to be evolutionarily neutral (Figure S1A in Additional file 1). Based on these observations, we conclude that the quality and genetic diversity of the DGRP make it suitable for global analyses of TFBS variation and these data are unlikely to elicit a prohibitive bias.
