To generate whole-genome multiple alignments, we first aligned the reference genome to each target genome in a pairwise manner. The process of pairwise whole-genome alignment consists of using Pattern-Hunter [105] to identify anchors of local alignment, grouping collinear anchors separated by a limited distance into chains, filtering out smaller chains that shadow larger ones, and finally using LAGAN [106] to globally align the entire chain. Once all genomes have been aligned to the reference, we then identified intervals of the reference that map tightly to a single interval of some or all of the target genomes, and we consider these the endpoints of blocks of multiple alignment. These blocks are generally smaller than any precursor pairwise alignment, because a rearrangement or loss of detectable similarity in any genome will truncate the block for all member genomes. We then ran the multiple aligner MLAGAN on each block. Finally, to facilitate searches for constrained regions of the reference, we projected the blocks onto the reference genome, effectively unwinding all genome rearrangements in the target genomes relative to the reference. We visualized the alignments in the Geno-meView browser [64] .