*The guide tree defines the alignment order, with branch lengths as substitutions per site. Rooted trees with branch lengths are preferred; if no branch lengths are provided, fixed lengths are used (see also Substitution scoring in Advanced alignment options). Unrooted trees with branch lengths are used with mid-point rooting. If no tree is provided, an approximate tree is computed using either Clustalw2 or PRANK.

Trust insertions (+F) is generally beneficial but may cause an excess of gaps if the guide tree is incorrect. Compute reliability is time consuming and may be disabled if not needed. This prevents some alignment post-processing options, however.

In addition to translation and alignment as codons or proteins, DNA sequences can be aligned using structure models. Fast/Slow assumes two types of sequence regions; Genomic additionally distinguishes coding exons.

Gaps are modelled as a time-dependent process and are more probable between distantly- than closely-related sequences. The probability of opening gaps is computed from gap rate and the pairwise distance between two sequences (or ancestral nodes) as defined in the guide tree. Gap length sets the mean for the geometrically-distributed expected gap lengths. Κ defines the ts/tv rate ratio for the HKY model that is used to compute the substitution scores for DNA alignments; the base frequences are empirical, based on the input data. Protein scoring is based on the WAG substitution model.

Substitution scoring is based on the branch lengths defined in the guide tree; option stringent sets the upper limit for maximum pairwise distance (or fixed branch length when none are provided) lower and thus enforces higher similarity. The alignment can be accelerated by using Clustalw2 for the generation of the first guide tree (when a structure model is not used, the alignment is iterated using a tree estimated from the first alignment) and allowing PRANK to use CHAOS-generated anchors for the alignment of long DNA sequences. The alignment with PRANK always infers the ancestral sequences at the internal nodes of the guide tree. These can be outputted and displayed in the alignment browser.

PRANK can also compute reliability and match stucture models on existing alignments. Alignment annotation works best for PRANK-generated alignments and using the original guide tree; it may work for other alignments too.

These options define the structure models for the alignment of DNA sequences. For them to have any effect you need to select one of the structure models in in Basic alignment options.

A structure model describes two or more processes and lets the aligner alternate between these during the alignment. The gap parameters and Κ are explained under Advanced alignment options. Length defines the length (mean of a geometric distribution) of fragments evolving under each process and the relative rate can set one of the processes to evolve either faster or slower.

Genomic model consists of five states, Fast, Slow and three Codon sites. Codon site processes are defined by the codon model and selection parameter Ω, with frequences optionally scaled by amino-acid frequencies. Length F+S defines the length of non-coding regions.