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Replicates of Rec-seq experiments with wild-kind Cre cluster together toward the center of the graph; Ala-substituted mutants that improve sequence choice appear to the left of the wild-sort grouping, whereas choice-diminishing variants cluster to the appropriate. Structural and mutagenesis studies23,26,31 advised that mutation of Arg259 would affect specificity at half-site position 10. Indeed, the Arg259→Ala variant confirmed a drop in enrichment at position 10 (from 5.0-fold for wild-sort Cre to 1.1-fold for the mutant), with a modest choice for C or T in the left half-site and G or A in the right half-site (Fig. 2b, c). Rec-seq revealed the sequence choice of Cre to be asymmetric, as is clear when the left and right half-site enrichment profiles are superimposed (Supplementary Fig. 6a). To ensure that an asymmetric sequence desire is a property of the enzyme and never due to the completely different DNA sequences flanking the library oligonucleotides (Fig. 1b), we performed Rec-seq using a substrate library similar to the original besides that the non-palindromic loxP core was changed with its reverse complement (Supplementary Fig. 6b). The Rec-seq enrichment profile of this "inverted core" loxP library mirrored, rather than duplicated, the profile on the original substrate library (Supplementary Fig. 6b), indicating that the oligonucleotide sequence context was not chargeable for the asymmetry of the Cre specificity profile

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To visually characterize one such map, we used the t-SNE algorithm37 to correlate the results of individual Rec-seq experiments utilizing multi-dimensional similarity analysis (Fig. 2g). The proximity of experiments within the t-SNE visualization relates their similarity across the total Rec-seq profile. Heat map of Rec-seq enrichment values for the CreR259A variant displaying the log2 of the enrichment value for each nucleotide at every position in loxP relative to the canonical base (black define). To characterize these interactions, we constructed 14 Cre mutants with Ala substitutions at residues identified to make contacts with loxP (Fig. 2a), purified each variant, and performed Rec-seq to map the functional relationship between particular residues and the DNA sequence preferences of Cre. Systemic change is required to ensure sentences are being commuted fairly and efficiently. Most Cre:loxP interactions are thought to involve the half-sites8,22, and we noticed minimal desire among the core nucleotides in experiments during which the half-websites had been held constant and the core was mutagenized (Supplementary Fig. 2). Finally, Rec-seq only captures mutations present in L1 and R2, as a result of the product of recombination containing R1 and L2 is degraded (Fig. 1b). With a view to isolate interactions between Cre and a single loxP half-site, solely L1 or R2 was randomized while R1 and L2 had been mounted as the wild-sort loxP sequence

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