The alignment matrices were analyzed by the maximum parsimony (MP) using PAUP* version 4.0b10 (Swofford 2003) with tree-bisection-reconnection (TBR) branch swapping and the MULTREES (holding multiple trees) option in effect with 1000 replicates of random sequence addition.
Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L).
The most parsimonious trees were obtained with heuristic searches of 1,000 replicates with random stepwise sequences addition, tree bisection-reconnection (TBR) branch swapping, collapse of zero-length branches, with the multiple-tree option in effect, and saving up to 10,000 trees from each random sequence addition.
This is distinct from progressive alignment methods because the alignment of prior sequences is updated at each new sequence addition.
Nodal support was estimated with a bootstrap procedure with 100 replications and 10 replicate trials of sequence addition.
We conducted 100 random sequence addition replicates under both clustering-constrained and unconstrained conditions and both ML and MP.
Heuristic ML searches were performed with 100 replicates of random sequence addition and tree bisection reconnection (TBR) branch swapping.
Per replicate, 10 independent random sequence addition runs followed by TBR branch swapping were conducted, saving only a single most parsimonious tree per run.
Bootstrap support was estimated from 1,000 replicates of simple sequence addition (parsimony) or as-is addition (likelihood) and TBR branch swapping.
Maximum parsimony(MP) analyses were conducted using PAUP version 4.0b10 [29] on G5 Macintosh computers by heuristic search and tree bisection reconnection (TBR) branch swapping with 1000 search replicates, each replicate with random sequence addition.
Under the default branch collapsing rule and simple sequence addition the number of equally parsimonious trees was very high (hitting the limit of 637.000 defined by memory allocation) and analyses without an upper limit for the number of saved trees could not be run to completion.
