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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
Molecular data can be imported from files of NBRF, FASTA, PHYLIP, CLUSTAL, and simple table format. It can also be exported to some of these
formats.
The Character Matrix Editor
can be used to edit a molecular sequence matrix. Standard ambiguity
codes are allowed.
Alter/Transform Tools
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 (some of these are available under "Other Choices"):
- Reverse Complement (DNA matrix only) —
reverses the order of contiguously selected blocks of sequence
and complements the sequence
- Collapse Gaps — collapses gaps in the selected block by pushing all sites to the left, to to yield
unaligned sequences
- Remove Gaps-Only Characters — Removes from the matrix all characters that consist of nothing but gaps.
- Shift Other to Match — Shifts other sequences to match a region in a selected sequences; described in the manual for the Align package.
- Convert Uncertainties to Polymorphisms — Converts all uncertainties in the selection to polymorphisms. For example, for a nucleotide sequence, a Y (C or T) will be converted into "C and T".
- Convert Polymorphisms to Uncertainties — Converts all polymorphisms in the selection to uncertainty. For example, for a nucleotide sequence, a cell that has both C and T will be converted into "C or T", i.e., Y.
- Arbitrarily Resolve State Ambiguity — Any cell that has partial uncertainty in the state (e.g., "Y", which is C or T) will be resolved into one of its states, chosen randomly. Thus, a Y will be converted to a C with 50% probability, and to a T with 50%.
- Nucleotide complement (DNA matrix only) —
enters the complementary sequence into the selected cells
- Trim Terminal Gap Characters — 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.
Alignment
The Align package contains utilities for sequence alignment. These include manual alignment tools (for shifting blocks of sequence, for example), and automated tools (e.g., sending a region of the matrix to ClustalW to align, or a pairwise alignment tool in the editor that will align one sequence to another).
See the Align manual for more details.
Finding Sequences
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).
Managing Molecular matrices
Managing sequences in different matrices, especially if from
different genes, can be difficult. Several functions assist in
this. These features are not restricted to molecular data, but
we anticipate most of their use will be with sequences.
- Include Taxa from
File: In the Taxa&Trees menu, this function reads
in an external file and attempts to concatenate taxa from the
file onto taxa blocks of the current file, and also to add their
data to existing matrices. This is useful to add new gene sequences
to an existing file. File formats that can be incorporated are
NEXUS, NBRF, and FASTA. This incorporation will NOT preserve
the associated information of the external NEXUS file such as
footnotes for the taxa and characters.
- Fused Matrix Export (NEXUS):
This is a special file exporter that can fuse matrices of different
genes as it exports. It is available by choosing Export in the
File menu. If you have a single taxa block
with multiple matrices attached to it (e.g., one for each gene),
then this exporter will write a single combined matrix with
the sequences of different genes concatenated for each taxon.
It will also write CHARPARTITION and CHARSETs to record which
section of the fused matrix corresponds to which source matrix.
It will also write a MrBayes block with that program's partitioning
commands. If the different matrices are of mixed type (DNA,
protein, non-molecular categorical) then you have a choice between
reducing them to simple categorical data or maintaining a mixed
matrix (suitable for MrBayes). Often however, you will have
slightly different names for the sequences of different genes,
and these may therefore exist in the file represented by different
taxa blocks. For instance, you may have a taxa block
with taxa "a.COI294", "b.COI815", and "c.COI290"
with corresponding matrix for COI sequences from species A,
B and C, and another taxa block with taxa "A98", "B312",
and "smith199628Sc" for 28S sequences from the same
species. The Fused Matrix exporter permits you to export these
into a single matrix as long as you have indicated how the sequences
correspond to one another. To do this, we suggest you create
a new taxa block representing the species or specimens. In this
example, create a taxa block "Species" with taxa A,
B and C. This will be the "master block of taxa" that
will organize the export. (Alternatively, you could choose one
of the genes' taxa blocks as the master block.) Set
up a Taxa Association between the master block of taxa and
each of the other blocks of taxa. With the first Taxa Association
between species and CO1 indicate that "a.COI294" belongs
with species A, "b.COI815" belongs with species B,
and "c.COI290" belongs with species C. Set up the
species-28S association similarly. Then when you choose Fused
Matrix Export, choose Species as your master taxa. The exporter
will then find all of the data corresponding to each species,
either under the species taxon itself or under one of the linked
taxa indicated by the Taxa Association, and compose a fused
matrix. If a single master taxon has more than one corresponding
taxa in one of the other matrices, the data are merged using
the same rules as for Merge
Taxa.
- Archiving Taxon Names:
In the List of Taxa window or the Character Matrix Editor, you
can use menu items in the Taxon Utilities submenu to archive
current taxon names in the file, and to recover archived versions
of the taxon names. You can see the archived names in the List
of Taxa window by choosing Columns>Archived
Name. Archiving names can be useful if you want to
temporarily rename taxa for export (e.g., shorter names for
use by CLUSTAL) and later recover the original names.
- Translation to protein: a new matrix of amino acids, derived by translation from existing protein-coding DNA matrix, can be formed by choosing Characters>Make New Matrix from>Translate DNA to Protein.
Display of Sequences
Protein-coding sequences can be colored by the amino acid into which a triplet would be translated (under the genetic code for that triplet) by choosing Matrix>Color Cells>Color Nucleotide by Amino Acid.
Genetic Codes
The genetic code for sequence data can be specified in Mesquite's
List of Characters window (by choosing List of Characters, and
then Columns>Current Genetic Codes, or with a data matrix frontmost,
Matrix>Genetic Codes...). Genetic codes are assigned to individual
characters (thus allowing one to have a mixed matrix of mitochondrial
and nuclear data, for example). To assign a genetic code, choose
select the characters, and use the popup menu of the title of
the "Genetic Code" column in the List of Characters
window.
The genetic code affects, among other things, the Translate DNA to Protein command, as well as the coloring of nucleotide sequences if Color Nucleotide by Amino Acid is chosen.
Interactions with GenBank
The following features allow one to interact with GenBank. They require that you have an Internet connection.
- BLAST in Web Browser: Select a sequence or portion thereof in the data matrix. Choose Matrix>Search>BLAST in Web Browser, and Mesquite will send a BLAST request to GenBank to search for matching sequences. Your default web browser should open and take you to the BLAST page.
- Top BLAST Matches: Select a sequence or portion thereof in the data matrix. Choose Matrix>Search>Top BLAST Matches, and you will be presented with a dialog box. In this you can choose:
- maximum number of matches to be sought by Mesquite
- whether the report of results should be saved to a text file
- whether details of the taxonomic lineage of the sequences should be reported
- whether to import the top matches into the current matrix
- the maximum time in seconds to wait for the BLAST search to complete.
- Fetch & Add GenBank Sequences: This option, in Matrix>Utilities> Fetch & Add GenBank Sequences, allows one to enter a comma-delimited list of GenBank accession numbers, and Mesquite will acquire these from GenBank and import them into the current matrix.
Simulating DNA sequence evolution
DNA sequence evolution can be simulated to build statistical
tests, for instance via parametric bootstrapping. See the page
on simulating DNA sequences.
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).
DNA Distances
Mesquite supports several distances for DNA data:
- Uncorrected (P) distances
- Jukes-Cantor
- Kimura 2-parameter
- F81 (Felsenstein, 1981)
- F84 (Felsenstein, 1984)
There are several options available (in the Distance Parameters submenu) for dealing with ambiguous bases and gaps:
- Count Sites with Gap in Pair: If this is chosen, then when the distance between two sequences is calculated, then sites that contain a gap in one of the sequences but not the other will be by default included in the calculation. If this option is unchecked, then any site at which either sequence is a gap will be excluded.
- Estimate Ambiguity Differences: If this is chosen, then sites in which one member of the pair has an ambiguity have their contribution to the distance estimated based upon the base patterns at non-ambiguous sites.
- Base Frequence on Entire Matrix: If this is chosen, then those distances that use as parameters the frequencies of bases (A, C, G, and T) will calculate those frequencies based on all sequences; if it is not chosen, then the frequencies are calculated based upon just the two sequences being compared at any one time.
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.
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