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Affinity maturation was then achieved by random addition of residues flanking the minimal binding sequence and by selection of new α-bungarotoxin binding peptides on the basis of their dissociation kinetic rate.
Global rearrangements were performed on 1000 starting trees produced by random addition of taxa.
MP trees were obtained using the Close-Neighbour-Interchange algorithm [77] with search level 3 [76], in which initial trees were obtained by random addition of sequences (10 replicates).
The Maximum Parsimony trees were calculated using the complete deletion option, all codon positions and a CNI level of 3 with an initial tree by random addition of sequences (10 replicates).
The MP tree was obtained using the close-neighbor-interchange algorithm with search level 1, in which the initial trees were obtained by random addition of sequences (10 replicates).
As a further investigation, starting with the final set of sparsified networks generated in the previous experiment, we re-densified their topologies by random addition of links and applied two approaches (ncMCE-R and FSW) at each percentage of densification.
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The Maximum Parsimony tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (page 126 in [ 21]) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates).
The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates).
Furthermore, MEGA was used to construct trees with the methods of neighbor joining (NJ) using different distance matrices, and maximum parsimony (MP) with heuristic searches using close-neighbor-interchang (CNI) level 3 and production of initial trees by of random addition of sequences (100 replicates).
Albergante et al. call this property Buffered Qualitative Stability (BQS): the network is qualitatively stable because its state does not change when the activity of particular regulatory links in the network changes, and it is buffered against its stability being compromised by the random addition of new links.
We used heuristic searches (10 replicates, random addition of taxa, with tree bisection and reconnection branch swapping), followed by 100 bootstrap iterations for robustness of the ML tree.
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