Analysis Options

In this dialog box you can select and view desired options in the Options Summary.  Options are organized in logical sections.  A yellow row indicates that you have a choice for that particular attribute. The primary sets of options available in this dialog box are:

Clock Settings

The RelTime analysis provides several options that are specific to divergence time estimation.

Clock Type

A RelTime analysis assumes that there is no global clock. You cannot change this parameter.

Clock Stringency

This selects how much separation clocks need to have before they can be merged. All Clocks keeps all of the clocks without merging any.  Many Clocks merges all clocks which are within 1 standard deviation of each other.  Few merges within 2 standard deviations, and Fewer merges within 3 standard deviations.

Variance Estimation Method

This offers the same options as the Phylogeny Test option in regular ML Tree construction (Bootstrap and Analytical).  For RelTime trees, MEGA will use the selected option to calculate the variance for estimated divergence times.

Phylogeny Test and Options

To assess the reliability of a phylogenetic tree, MEGA provides the Bootstrap test.  This uses the bootstrap re-sampling strategy, so you need to enter the number of replicates.

Substitution Model

In this set of options, you can choose various attributes of the substitution models for DNA and protein sequences.

Substitutions Type

Here you may select a substitutions type of Nucleotide, Syn-Nonsynonymous, or Amino Acid.  The selection in this row affects the available models in the model row.

Model

Here you select a stochastic modelHC_Models_for_estimating_distances for estimating evolutionary distance by clicking on the row then selecting a model for the current Substitutions Type. 

Substitutions to Include

Depending on the distance model or method selected, the evolutionary distance can be teased into two or more components.  By clicking on the row, you will be provided with a list of components relevant to the chosen model.

Transition A transition occurs when a purine is substituted by a purine, or a pyrimidine by a pyrimidine./Transversion A change from a purine to a pyrimidine, or vice versa, is a transversion. Ratio

This option will be visible if the chosen model requires you to provide a value for the Transition A transition occurs when a purine is substituted by a purine, or a pyrimidine by a pyrimidine./Transversion A change from a purine to a pyrimidine, or vice versa, is a transversion. ratio (R)RH_Transition_Transversion_Ratio_R.

Pattern among Lineages

This option becomes available if the selected model has formulas that allow the relaxation of the assumption of homogeneity of substitution patterns among lineages.

Rates among Sites

This option becomes available if the selected distance model has formulas that allow rate variation among sites.  If you choose gamma-distributed rates, then an RH_Gamma_parameoption to specify the number of discrete categories for modeling the gamma distribution becomes visible.

Data Subset to Use

These are options for handling gaps and missing data, including or excluding codon A codon is triplet of nucleotides that codes for a specific amino acid. positions, and restricting the analysis to labeled sites Sites in a sequence alignment can be categorized and labeled with user-defined symbols. Each category is represented by a letter or a number. Each site can be assigned to only one category, although any combination of categories can be selected for analysis. Labeled sites work independently of and in addition to genes and domains, thus allowing complex subsets of sites to be defined easily., if applicable.

Gaps and Missing Data

You may choose to remove all sites containing alignment gaps Phylogenetic analysis on two or more DNA or amino acid sequences requires that the sequences be aligned so that the substitutions can be accurately enumerated. During alignment, gaps must be introduced in sequences that have undergone deletions or insertions. These gaps are known as alignment gaps or indels. and missing information before the calculation begins (Complete-deletionRH_Complete_Deletion_Option option).  Alternatively, you may choose to retain all such sites initially, excluding them as necessary in the pairwise distance estimation (Pairwise-deletionRH_Pairwise_deletion_option option), or you may use Partial DeletionRH_Partial_Deletion_Option (Site coverage) as a percentage.

Codon A codon is triplet of nucleotides that codes for a specific amino acid. Positions

Check or uncheck the boxes for any combination of 1^st, 2^nd, 3^rd, and non-coding positions for analysis.  This option is available only if the nucleotide sequences contain protein-coding regions and you have selected a nucleotide-by-nucleotide analysis.

Labeled Sites Sites in a sequence alignment can be categorized and labeled with user-defined symbols. Each category is represented by a letter or a number. Each site can be assigned to only one category, although any combination of categories can be selected for analysis. Labeled sites work independently of and in addition to genes and domains, thus allowing complex subsets of sites to be defined easily.

This option is available only if some or all of the sites have associated labels.  By clicking on the row, you will be provided with the option of including sites with selected labels.  If you choose to include only labeled sites Sites in a sequence alignment can be categorized and labeled with user-defined symbols. Each category is represented by a letter or a number. Each site can be assigned to only one category, although any combination of categories can be selected for analysis. Labeled sites work independently of and in addition to genes and domains, thus allowing complex subsets of sites to be defined easily., then these sites will be the first extracted from the data.  Then all other options mentioned above will be enforced.  Note that labels associated with all three positions in the codon A codon is triplet of nucleotides that codes for a specific amino acid. must be included for a full codon A codon is triplet of nucleotides that codes for a specific amino acid. to be incorporated in the analysis.

Tree Inference Options

ML Heuristic Method

There are two options available Nearest-Neighbor-Interchange (NNI)Nearest_Neighbor_Interchange_NNI_ or Subtree-Pruning-Regrafting (SPR) For any tree searching method, exhaustive search, where all possible topologies are considered is unfeasible for even a small number of taxa. Subtree Pruning And Regrafting is a tree topology search heuristic which reduces the number of topologies searched by performing the following operations on the tree. First, a subtree of the current best tree is selected and detached (pruned). Second, the detached subtree is regrafted onto another branch of the remaining tree, in such a way that a new topology is created and then likelihood of the new topology is calculated. This procedure is repeated for all regrafting positions that produce new topologies using the pruned subtree. The procedure is also repeated for each subtree (within the designated search level) and if the topology with best likelihood among those scored gives sufficient improvement over the current best tree, that topology becomes the current best tree. This is repeated until no significant further likelihood improvements are obtained. A single pass of the SPR algorithm examines O(N2) new trees, where N is the number of leaves in the original tree. This is because, for each subtree there are O(N) possible regraftings, and there are O(N) possible subtrees to consider. In contrast, NNI examines O(N) topologies at each pass of the algorithm.Subtree_Pruning_Regrafting. For SPR, two search levels are available; search level 3, which searches less exhaustively and search level 5 which performs an extensive search of the tree space.

Initial Tree for ML

The initial tree for ML can be supplied in several ways. Mega can simply create it on the fly using Neighbor Joining (NJ), BioNJ, Maximum Parsimony, or try both NJ and BioNJ and select the superior tree or you may specify your own tree by providing a newick file.

Branch A branch is a line connecting either two internal nodes to each other or an external node to an internal node in a phylogenetic tree. The length of a branch denotes the genetic distance (e.g., number of substitutions per unit time) between the two taxa it connects. Swap This command changes the cursor to the 'Flip' icon. Then, you click on an interior branch, MEGA swaps the two subtrees defined by this branch. If each of the subtrees is an individual taxon, then Swap is the same as Flip. The cursor will revert to the arrow if you click on the arrow icon on the left-hand side of the Tree Explorer. Filter

For analyses that use a Maximum Likelihood approach, the ‘branch A branch is a line connecting either two internal nodes to each other or an external node to an internal node in a phylogenetic tree. The length of a branch denotes the genetic distance (e.g., number of substitutions per unit time) between the two taxa it connects. swap This command changes the cursor to the 'Flip' icon. Then, you click on an interior branch, MEGA swaps the two subtrees defined by this branch. If each of the subtrees is an individual taxon, then Swap is the same as Flip. The cursor will revert to the arrow if you click on the arrow icon on the left-hand side of the Tree Explorer. filter’ analysis setting is used to set the stringency of optimization with respect to branch A branch is a line connecting either two internal nodes to each other or an external node to an internal node in a phylogenetic tree. The length of a branch denotes the genetic distance (e.g., number of substitutions per unit time) between the two taxa it connects. lengths and improvements in log likelihood values. A weaker filter will result in more exhaustive optimization so that the search may take longer but potentially, a larger search space will be explored. A stronger filter will result in less exhaustive optimization so that the search may require less time but potentially, a smaller search space will be explored.

System Resource Usage

Number of Threads

Beginning in MEGA5.1, multithreaded ML phylogeny inference can be performed on computers that have multiple CPUs or cores. If MEGA detects multiple processing resources, an option for setting the number of search threads will be provided in the analysis preferences dialog. However, this option is only available when running a single ML inference analysis. If you wish to run multiple ML based analyses simultaneously, set the number of threads to ‘1’ when initializing the first analysis.