Molecular data

Molecular data (DNA or protein sequences) can be edited, manipulated, simulated and analyzed in various ways in Mesquite. Most of the features discussed elsewhere concerning editing and analysis of general categorical data also apply to molecular data; here we focus on features specifically designed for sequence data.

Contents

• Editing molecular data
• Simulating DNA sequence evolution
• Statistics for DNA sequences
• Statistics for Protein sequences
• Visualizing tertiary structure
• Sequence Data within populations
• Reconstructing ancestral states

Editing molecular data

Molecular data can be imported from files of NBRF format, PHYLIP format, and simple table format. It can also be exported to these formats.

The Character Matrix Editor can be used to edit a molecular sequence matrix. Standard ambiguity codes are allowed.

The following can be applied to all or the selected portions of a molecular sequence matrix in the Character Matrix Editor. These are available under the Alter/Transform submenu of the Matrix menu:

• Nucleotide complement (DNA matrix only) — enters the complementar sequence into the selected cells
• Reverse sequence — reverses the order of contiguously selected blocks of sequence
• Collapse Gaps — collapses gaps to yield unaligned sequences
• Collapse Gaps-Only Edges — deletes characters at edges of matrix that are gaps-only.

Other options may appear; see the page on characters for standard choices in this submenu. You can also apply the other editing tools described for character matrices.

The view of the matrix can be adjusted in various ways. Cells can be colored according to the state at the site (Color Cells submenu, Character State) or according to a value like the GC bias (Color Cells submenu, Cell Value; can request this coloring to use a moving window). Examples of this are shown below. The Display submenu of the Matrix menu contains other options such as a Bird's eye view which makes the cells narrow to show more of the sequences.

Copy Sequence (at bottom of Edit menu) copies the selected cells of the matrix into the computer's clipboard as a sequence. That is, whereas the standard Copy would place into the clipboard selected pieces of the matrix in tab-delimited text format (e.g., if the sequence AATCA is selected, "A-tab-A-tab-T-tab-C-tab-A" would be copied), this modified Copy Sequence command does not include tabs (thus, "AATCA" would be copied). This style of copying is useful when interacting with programs like Sequencher (TM). For instance, if you want to find a piece of sequence in a matrix in Mesquite within a chromatogram viewer of Sequencher, do the following: select the sequence in Mesquite, choose Copy Sequence, then go to Sequencher, select Find Bases, and paste the sequence as the search string.

Pieces of sequences can be found using the Find Sequence and Find All Sequences submenus of the Edit menu. The current options are:

• Matching Sequence: This finds sequences matching a target sequence the user enters. The search allows a certain number of mismatches. Optionally, it can search for the reverse, complement and reverse complement of the target sequence.
• Maintain Target Match: This highlights and maintains highlighted the first occurence of a given sequence in a given taxon. First, you are asked which taxon to search in. Then, it displays a panel like this:
  
  underneath the matrix. The first button (red X) is to close the panel; the second pauses the search; the third allows you to select another taxon as your focus. If you type a sequence into the text area, the matching sequence (if any) will be highlighted in the matrix. Mesquite is constantly monitoring this text, and so you don't need to give any command to find again if you change the text. This is useful if working with a program like Sequencher. If you see a stretch of sequence while viewing chromatograms that you'd like to find in the matrix in Mesquite, type in the sequence into the text box and you will quickly be taken to it in the taxon.
• Maintain Clipboard Match: This is similar to Maintain Target Match, except that it obtains the search string not from the text area but from the clipboard. If the clipboard changes, the function will automatically find the sequence again in the matrix. This is useful if working with a program like Sequencher. If you turn on Maintain Clipboard Match, then you can copy stretches of a sequence within Sequencher, and Mesquite will automatically highlight it, without your having to return to Mesquite or give any other command to it. (Mesquite is constantly monitoring the clipboard to see if it changes).

Statistics for DNA sequences

Calculations for categorical characters in general can be applied to DNA sequences. For example, Parsimony calculations can be made for DNA sequences, as can basic descriptive statistics such as the percent of a sequence or character that is missing data or gaps. In addition, there are several modules specifically designed for DNA data, illustrated by examples in Mesquite_Folder/examples/Molecular. These calculate compositional bias:

• ACGT Compositional Bias — This module supplies the compositional bias of taxa, measured over the taxon's sequence. The bias is treated as a continuous character, and thus can be used wherever characters are used, as for instance in the reconstruction of the evolution of compositional bias as shown in the image below. It can return either the proportion G+C, or separately A, C, G, and T proportions.

• Character Compositional Bias — This module supplies the compositional bias for characters. It calculates the percent of taxa with particular nucleotides (GC bias, or individual frequency of A, C, G or T) for a character. The image below shows a moving window analysis of compositional bias along a sequence; the instructions for generating the chart are given here.

• GC bias coloring of matrices — The cells of the Character Matrix Editor may be colored according to a moving window of GC bias along the sequence, as shown below, by selecting Matrix>Color Cells>Color By Cell Value, then once shown the colors can be smoothed by a moving window analysis by selecting Matrix>Moving Window (for colors).

Statistics for Protein Data

• Site hydrophobicity — This module supplies the average amino acid hydrophobicity, averaged across taxa, for each site. It can be used in charts, for instance to see the relationship between a phylogenetic statistic for the site (character) and it average hydrophobicity. This chart, for example, shows parsimony character steps as a function of hydrophobicity:

• Amino Acid hydrophobicity — The cells of the Character Matrix Editor may be colored according to a moving window of hydrophobicity along the sequence, as shown below, by selecting Matrix>Color Cells>Color By Cell Value, then once shown the colors can be smoothed by a moving window analysis by selecting Matrix>Moving Window (for colors).

Visualizing tertiary structure

Although there are not yet dedicated windows for visualizing phylogenetic statistics in the context of molecular structure, features have been added to the Scattergram chart to allow it to be adapted for this purpose. For instance, in this image cytochrome B is shown, with the amino acids colored according to a simple phylogenetic statistic: the number of parsimony steps on a phylogeny. The colors are smoothed by a moving window, and show that several coils of the molecule, a few at the left and one deep at the right, evolve more rapidly than others. This example is illustrated in the data file at Mesquite_Folder/examples/Molecular/06-cytochromeB.nex

To build such a chart, begin with a file with a matrix of protein sequences. The procedure is also described in the example files 08-cytochromeBlinked.nex and 09-cytochromeBscatter.nex.

• Select New Linked Matrix from the Characters menu. When a matrix is made to be linked to a second matrix, the two matrices are constrained to have the same number of characters.
• Indicate that you want the linked matrix to be a Continuous matrix, and link it to your protein matrix. Then, turn it into a three dimensional matrix (Taxa X Characters X Coordinates [x, y and z]) by using Add Item and Rename Item in the Utilities submenu of the Matrix menu of the Character Matrix Editor. The x,y,z coordinates could be added for all taxa if known, but otherwise only one taxon needs to be filled out (because we will use the average x,y,z coordinates for the amino acids).
• Once the linked matrix of xyz amino acid positions is entered, select Analysis>New Scattergram for> Characters. Indicate you want the scattergram to be for Stored Characters, and indicate Same value for the two axes. In the dialog box "Values for axes", choose Mean Value of Character (Linked Matrix). In response to "Use characters from which matrix? (for Character Source)" choose the protein sequence matrix as the matrix to be used. This will plot the sites (amino acids, characters) in their correct places, but as a series of round spots.
• To change the appearance of the plot, select Join the Dots in the Special Effects submenu of the Scattergram menu. Then select Thick Joints, deselect Show Dots, deselect Join First to Last, and set the marker size larger (e.g., 8). This will result in a plot as shown above, but without the colors.
• Next, choose Color by Third Value from the Colors menu and choose the value by which to color the amino acids. For parsimony steps, for instance, choose Character Value with current tree.
• Finally, to use a moving window to smooth the colors, select Moving Window for Colors from the Colors menu and indicate the window size (e.g., 5).

Sequence data within populations

See the page on population genetics.

Reconstructing ancestral states

Ancestral states of continuous characters can be reconstructed as described in the page on reconstructing ancestral states. Likelihood methods are not yet available for molecular characters.