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TutorialGuide to Notations Used
Introduction to Walk through MEGA
This walk-through provides several brief tutorials that explain how to perform common tasks in MEGA. Each tutorial requires the use of sample data files which can be found in the /MEGA/Examples folder (default location for Windows users is C:\Program Files\MEGA\Examples\. The location for Mac users is $HOME/MEGA/Examples, where $HOME is the user’s home directory). It is recommended that you follow the examples for a given tutorial in the order presented as the techniques explained in the initial examples are used again in the subsequent ones. In the tutorials, the following conventions are used: · Keystrokes are indicated by bold letters (e.g., F4). · If two keys must be pressed simultaneously, they are shown with a + sign between them (e.g., Alt + F3 means that the Alt and F3 keys should be pressed at the same time). · Italicized words indicate the name of a menu or window. · Italicized bold words indicate individual commands that are found in menus, submenus, and toolbars. ·
‘Main menu’ refers to the menu bar at the top of the
currently active window (File, Analysis, Help, etc.). ·
‘Main
MEGA menu’ refers to the menu on the main window of MEGA where you launch all
of the analyses from. ·
‘Launch
bar’ refers to the toolbar located directly below the main menu of the
currently active window (Align, Data, Models, Distance, etc.). ·
For
brevity, a sequence of menu / button clicks is indicated by a sequence of
commands separated by pipes (e.g., ‘File | Open’ indicates that you should
click on the ‘File’ main menu item and then click on the ‘Open’ sub menu item
that is displayed). I want to learn
about: 1. Mega Basics 3.
Estimating Evolutionary Distances
4.
Building Trees from Sequence Data 6.
Working with Genes and Domains 9.
Computing Sequence Statistics 10. Building Trees from
Distance Data 11. Constructing
Likelihood Trees Aligning Sequences
In this tutorial, we will show
how to create a multiple sequence alignment from protein sequence data that
will be imported into the alignment editor using different methods. All of the data files used in this tutorial
can be found in the MEGA\Examples\
folder (The default location for Windows users is C:\Program Files\MEGA\Examples\. The location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Opening
an Alignment The Alignment Explorer is the tool for building and editing multiple
sequence alignments in MEGA. Example 2.1: Launch the Alignment
Explorer by selecting the Align |
Edit/Build Alignment on the launch bar of the main MEGA window. Select Create New Alignment and click Ok.
A dialog will appear asking “Are you building a DNA or Protein sequence
alignment?” Click the button labeled “DNA”. From the Alignment Explorer main menu, select Data
| Open | Retrieve sequences from File. Select the "hsp20.fas"
file from the MEG/Examples directory. Aligning Sequences
by ClustalW You can create a multiple sequence alignment in MEGA using either the
ClustalW or Muscle algorithms. Here we align a set of sequences using the
ClustalW option. Example 2.2: Select the Edit
| Select All menu command to select all sites for every sequence in the
data set. Select Alignment
| Align by ClustalW from the main menu to align the selected sequences
data using the ClustalW algorithm. Click the “Ok” button to accept the
default settings for ClustalW. Once the
alignment is complete, save
the current alignment session by selecting Data
| Save Session from the
main menu.
Give the file an appropriate name, such as "hsp20_Test.mas". This
will allow the current alignment session to be restored for future editing. Exit the Alignment Explorer by selecting Data | Exit Aln Explorer from
the main menu. Aligning
Sequences Using Muscle Here we describe how to create a multiple sequence alignment using the
Muscle option. Example 2.3: Starting from the main MEGA window, select
Align
| Edit/Build Alignment from the launch bar. Select Create a new alignment and then select DNA. From the Alignment
Explorer window, select Data | Open | Retrieve sequences from a file
and select the “Chloroplast_Martin.meg” file from the MEGA/Examples directory. On the Alignment
Explorer main menu, select Edit | Select All. On the Alignment
Explorer launch bar, you will find an icon that looks like a flexing arm.
Click on it and select Align DNA. Near the bottom of the MUSCLE - AppLink window, you will see a row called Alignment Info. You can scroll through
the text to read information about the Muscle program. Click on the Compute button (accept the default
settings). A Progress window will
keep you informed of Muscle alignment status. In this window, you can click on
the Command Line Output tab to see
the command-line parameters which were passed to the Muscle program. Note: The analysis may complete so fast, that
you won’t be able to click on this tab or read it. The information in this tab isn’t essential,
it’s just interesting. When the Muscle program has finished, the aligned
sequences will be passed back to MEGA and displayed in the Alignment Explorer window. Close the Alignment
Explorer by selecting Data | Exit Aln Explorer. Select No
when asked if you would like to save the current alignment session to file. Obtaining
Sequence Data from the Internet (GenBank) Using MEGA’s integrated browser you can fetch GenBank sequence data from
the NCBI website if you have an active internet connection. Example 2.4: From the main MEGA window, select Align | Edit/Build Alignment from the main menu. When prompted, select Create New Alignment and
click ok. Select DNA Activate MEGA’s integrated browser by selecting Web | Query Genbank from the main
menu. When the NCBI: Nucleotide site is loaded, enter
CFS as a search term into the search
box at the top of the screen. Press the Search button. When the search results are displayed, check the box
next to any item(s) you wish to import into MEGA. If you have
checked one box:
Locate the dropdown menu labeled Display
Settings (located near the top left hand side of the page directly
under the tab headings). Change its value to FASTA and then click Apply.
The page will reload with all the search results in a FASTA format If you have
checked more than one box: locate the Display
Settings dropdown (located near the top left hand side of the page
directly under the tab headings). Change the value to FASTA (Text) and click
the Apply button. This
will output all the sequences you selected as a text in the FASTA format. Press the Add to Alignment button (with the
red + sign) located above the web
address bar. This will import the sequences into the Alignment Explorer. With the data now displayed in the Alignment
Explorer, you can close the Web
Browser window. Align the new data using the steps detailed in the
previous examples. Close the Alignment
Explorer window by clicking Data | Exit Aln Explorer. Select No
when asked if you would like the save the current alignment session to file. Note: We have aligned some sequences and they are now ready to be analyzed.
Whenever you need to edit/change your sequence data, you will need to open it
in the Alignment Editor and edit or
align it there. Then export it to the MEGA format and open the resulting file. Estimating Evolutionary Distances
In this tutorial, we will estimate evolutionary
distances for sequences from 11 Drosophila species using various models.
The data files used in this tutorial can be found in the MEGA/Examples folder (The default location for Windows users is C:\Program Files\MEGA\Examples. The default
location for Mac users is $HOME/MEGA/Examples,
where $HOME is the user’s home directory). Estimating
Evolutionary Distances Using Pairwise Distance In MEGA, you can estimate evolutionary distances
between sequences by computing the proportion of nucleotide differences between
each pair of sequences. Example
3.1: Open the "Drosophila_Adh.meg" data file. If
needed, refer to the “MEGA Basics” tutorial. From the main MEGA launch bar, select Distance | Compute Pairwise Distance. In the Analysis
Preferences window, click the Substitutions Type pull-down and then
select the Nucleotide option. Click the pull-down for Model/Method and
select the p-distance model. For this example we will be using the
defaults for the remaining options. Click Compute to begin the computation. A progress indicator will appear briefly and then
the distance computation results will be displayed in grid form in a new
window. Leave this window open so we can compare the results from the next
steps. Compute and
Compare Distances Using Other Models/Methods MEGA supports a wide collection of models for estimating evolutionary
distances. Here we compare evolutionary distances calculated by using different
models. Example
3.2: Repeat Example 3.1 above, but select the Jukes/Cantor
model under the Model/Method
pull-down instead of the p-distance model, leaving all the
other options the same. Again, leave the results window open for comparison. Repeat the analysis, this time selecting the Tamura-Nei
model under the Model/Method
pull-down, leaving all the other options the same. Again, leave the results
window open for comparison. You are now able to compare the three open result
windows which contain the distances estimated by the different methods. After you have compared the results, select the File | Quit Viewer option
for each result window. Do not close the "Drosophila_Adh.meg"
data file. Compute
the Proportion of Amino Acid Differences You can also calculate
evolutionary distances based on the proportion of amino acid differences. Note:
MEGA
will automatically translate nucleotide sequences into amino acid sequences
using the selected genetic code table. The genetic code table can be edited by Data | Select Genetic Code Table
from the main MEGA launch bar. Example 3.3: From the main MEGA window, select Distance | Compute Pairwise Distances
from the main menu. This will display the Analysis Preferences window. Click the Substitutions Type pull-down,
select Amino Acid and then select p-distance under Model/Method. Click the Compute
button to accept the default values for the rest of the options and begin the
computation. A progress dialog box will
appear briefly. As with the nucleotide estimation, a results viewer window will
be displayed, showing the distances in a grid format. After you have inspected the results, use the File | Quit Viewer command to
close the results viewer. Close the data by selecting the Close
Data button on the main MEGA task bar. Building Trees from Sequence Data
In this tutorial, we will illustrate the procedures for
building trees and in-memory sequence data editing, using the commands available
in the Data and Phylogeny
menus. We will be using the "Crab_rRNA.meg" file which can be found
in the MEGA/Examples directory. This file contains nucleotide sequences for the
large subunit mitochondrial rRNA gene from different crab species (Cunningham et
al. 1992). Since the rRNA gene is transcribed, but not translated, it falls
in the category of non-coding genes. The “Crab_rRNA.meg” file used in this tutorial can be found in the MEGA/Examples folder (The default
location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Building a
Neighbor-Joining (NJ) Tree In this example,
we will illustrate the basics of phylogenetic tree re-construction using MEGA
and become familiar with the Tree
Explorer window. Example 4.1: Activate the "Crab_rRNA.meg" data file. If
necessary, refer to Example 1.2 of the “MEGA Basics” tutorial. From the main MEGA launch bar, select Phylogeny | Construct/Test Neighbor-Joining Tree
menu option. In the Analysis Preferences window select the
p-distance
option from the Model/Method drop-down. Click Compute to accept the defaults for
the rest of the options and begin the computation. A progress indicator will
appear briefly before the tree displays in the Tree Explorer window. To select a branch, click on it with the left mouse
button. If you click on a branch with the right mouse button, you will get a
small options menu that will let you flip the branch and perform various other
operations on it. Select a branch and then press the Up, Down, Left, and Right arrow keys to see how the cursor
moves through the tree. Change the branch style by selecting the View | Tree/Branch Style command
from the Tree Explorer main menu. Select the View
| Topology Only command from the Tree Explorer main menu to
display the branching pattern on the screen. You can display the
numerical branch lengths in the Topology Only option by selecting View | Options and clicking on the Branch tab. Check the box labeled Display Branch Length and click Ok. Printing the NJ
Tree (For Windows users) Windows users can print directly from Tree
Explorer. Example 4.2a: Select the File
| Print option from the Tree Explorer main menu to bring up a
standard Print window. This will
print the tree full-sized and may take multiple sheets of paper. Press Cancel. To restrict the size of the printed tree to a single
sheet of paper, choose the File |
Print in a Sheet command from the Tree Explorer main menu. Press
Ok. Select the File
| Exit Tree Explorer command
to exit the Tree Explorer. Click the OK button to close the Tree
Explorer without saving the tree session. Printing the NJ
Tree (For Mac users) MEGA does not support printing directly from Tree Explorer when running on a Mac system. To print a tree using a
Mac, users can save the tree image to a PDF file and then print it by normal
means. Example 4.2b: Select the Image
| Save as PDF File option from the Tree Explorer main menu to
bring up a standard Save
window. Save the image to the desired location. Once the document is saved, you can open it with
your PDF reader and print the document in the same manner as any other PDF
document. Select the File
| Exit Tree Explorer command
to exit the Tree Explorer. Click the OK button to close the Tree
Explorer without saving the tree session. Construct a
Maximum Parsimony (MP) Tree Using the Branch-&-Bound Search Option Using MEGA, you can re-construct a phylogeny using Maximum Likelihood,
Minimum Evolution, UPGMA, and Maximum Parsimony methods in addition to
Neighbor-Joining. Here we re-construct the phylogeny for the “Crab_rRNA.meg”
data using the Maximum Parsimony (MP) method. Example 4.3 Select the Phylogeny | Construct/Test Maximum Parsimony
Tree(s) menu option from the main MEGA launch bar. In the Analysis
Preferences window, choose Max-mini Branch-&-bound for the MP
Search Method option. Click the Compute button to accept the
defaults for the other options and begin the calculation. A progress window
will appear briefly, and the tree will be displayed in Tree Explorer. (Windows users) Now print this tree by selecting
either of the Print options
from the Tree Explorer's File menu. (Mac users) Save the tree
to a PDF file as described in Example 4.2b above. Compare the NJ and MP trees. For this data set, the
branching pattern of these two trees is identical. Select the File
| Exit Tree Explorer command
to exit the Tree Explorer. Click OK to close Tree Explorer
without saving the tree session. Constructing a
MP Tree using the Heuristic Search For each method of phylogenetic inference, MEGA provides numerous
options. In this example, we conduct MP analysis using the Min-Mini Heuristic
search. Example 4.4: Follow the steps in Example
4.3 and instead of choosing Max-mini
Branch-&-bound, choose Min-Mini
Heuristic for MP Search Method.
Change the MP Search Level to 2 and click Compute. Note: In this example, the same tree is obtained by the Max-mini Branch-&-bound option as in the Min-Mini Heuristic option as long as the MP Search Level is set to 2.
However, the computational time is much shorter for the Heuristic method. Examining Data
Editing Features For noncoding sequence data, OTUs (Operational Taxonomic Units) as well
as sites can be selected for analysis. Example 4.5: From the main MEGA window select the Data | Select Taxa and Groups
option from the launch bar. A dialog box is displayed. All the OTU labels are checked in the left panel.
This indicates that all OTUs are included in the current active data subset. To
remove the first OTU from the data, uncheck the checkbox next to the first OTU
name in the left panel. Click the Close button. Now, when you construct a neighbor-joining tree from
this data set, it will contain 12 OTUs instead of 13. Close out of the Tree Explorer window by selecting File
| Exit Tree Explorer and do not save. Deactivate the operational data
set by selecting the Close Data icon from the main
MEGA window. Testing Tree Reliability
In this example, we will conduct
two different tests of reliability using protein-coding genes from the
chloroplast genomes of nine different species. The data file “Chloroplast_Martin.meg” which is used in this tutorial can
be found in the MEGA/Examples folder (The default
location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Bootstrap
Testing for a Neighbor-Joining Tree Example 5.1: Activate the "Chloroplast_Martin.meg" file.
If necessary, refer to Example 1.2 of “MEGA Basics”. On the main MEGA window task bar, select the Phylogeny | Construct/Test
Neighbor-Joining Tree option. The Analysis Preferences window appears on
the screen. For the Model/Method, select p-distance. Select Bootstrap
method for the Test of Phylogeny option. Click Compute to accept the default values
for the rest of the options. A progress indicator provides the progress of the
test as well as the details of your analysis preferences. Once the computation is complete, the Tree
Explorer appears and displays two tree tabs. The first tab is the original tree and the second is the Bootstrap consensus tree. To produce a condensed tree, use the Compute | Condensed Tree main menu command from the Tree
Explorer window. You can further manipulate the appearance of the condensed
tree here. To change the cutoff value, select the View | Options menu command and click the Cutoff tab. For
now, keep the Cut-off value at 50% and click the OK button. This tree shows all the branches that are supported
at the default cutoff value of BCL ³
50. Select the Compute | Condensed
Tree main menu
command and the original NJ tree will reappear. From the Tree Explorer window, select the Image | Save as PDF File option and
save a PDF image of the tree to a convenient location. From the Tree Explorer window, select the File | Exit Tree Explorer command
to exit the Tree Explorer. A warning box will inform you that your tree data
has not been saved. Click Ok to close Tree Explorer
without saving the tree. Interior-branch testing for the
Neighbor-Joining Tree For neighbor-joining trees, you may conduct the standard error test for
every interior branch by using the Interior branch test of phylogeny. Example 5.2: From the main MEGA window, select Phylogeny | Construct/Test
Neighbor-Joining Tree from the launch bar. In the Analysis Preferences dialog, make sure
the Substitutions Type option is set to Amino Acid and the Model/Method
is set to p-distance. Set the Test of Phylogeny option to Interior-branch
test. Click Compute to begin the computation. A
progress indicator window will appear briefly. When the tree appears, confidence
probabilities (CP) from the standard error test of branch lengths are displayed
on the screen. Compare the CP values on this tree with the BCL
values of the tree that you saved as a PDF file in the previous exercise. Now close the Tree Explorer by selecting File
| Exit Tree Explorer from the main menu. Close the current data by
clicking the Close Data icon on the main MEGA window. Working With Genes and Domains
Defining and
Editing Gene and Domain Definitions In this example we will demonstrate how to specify coding and non-coding
regions of a sequence. We will be using the file “Contigs.meg” which is located
in the MEGA/Examples directory folder (The default location for Windows users
is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the user’s home directory). Example 6.1: Activate the data file "Contigs.meg". If
necessary, refer to Example 1.2 of the “MEGA Basics” tutorial. From the main MEGA window launch bar, select Data | Select Genes and Domains. Notice the column header bar across the top (‘Name’, ‘From’,
‘To’, ‘#Sites’, ‘Coding?’ 'Codon Start’). Domains
will be listed under the column header labeled ‘Name’. Click on the domain labeled Data underneath the Genes/Domains group, then click on the
button labeled Delete/Edit. Select Delete Gene/Domain to delete the data domain. Click on the Genes/Domains label and then click
the Add
Domain button. Select Add New Domain from the popup menu. Right-click on the new domain and select
Edit
Name from the popup menu. Change the name to “Exon1” and press
the Enter key. Select the ellipses (…) button next to the first question mark in the ‘From’ column to set the first site of
the domain. When the Start site for Exon1
window appears, select site number 1
for the AC087512 chimp row and push
the Ok
button. Select the ellipsis (…) button in the ‘To’
column to set the last site of the domain. When the End site for Exon1 window appears, select site number 3918 for the AC087512 chimp row and push the OK button. Check the box in the ‘Coding?’ column to
indicate that this domain is protein coding. You will need to click the box
three times before the check mark appears. Add two more domains to the Genes/Domains
item using the same steps. One of these domains will be named “Intron1” and
will begin at site 3919 and end at
site 5191. The other will be named “Exon2”
and will begin at site 5192 and end
at site 8421. Be sure to check the
checkbox in the ‘Coding?’ column for Exon2 to indicate a protein-coding
domain. Click on the Genes/Domains
item to highlight it and then click the Add Gene button at the bottom of the
screen. From the popup menu choose Add new gene at the end. Right click
on this new gene and change the name to “Predicted Gene”. Click and drag all of
the newly created domains to the Predicted
Gene so that they now appear under the new gene. Press the Close button at the bottom of the
window to exit the Gene/Domain Organization window. Using Domain
Definitions to Compute Pairwise Distances Now, if we compute pairwise distances between our sequences, the
non-coding regions that we specified in the example above will be ignored. Example 6.2: From the main MEGA window, select the Distance | Compute Pairwise Distances
option from the launch bar. In the Analysis Preferences window, click on
the Substitutions Type drop-down and select Nucleotide. The Select
Codon Positions row is now enabled. Make sure that the Noncoding sites option does not have a checkmark next to it. Click
the Compute
button to begin the analysis. When the computation is
complete, the Pairwise Distances
window will display the pairwise distance computed using only the
sequence data from exonic domains of the Predicted
Gene.
Close the Pairwise Distances window by selecting File | Quit Viewer and
the Sequence Data Explorer window by selecting the Close Data icon on the
main MEGA window. Testing for Selection
In this example, we describe how
to perform a codon-based test of positive selection for five alleles from the
human HLA-A locus (Nei and Hughes 1991). The “HLA-3Seq.meg" data file, which is used in this tutorial,
can be found in the MEGA/Examples
folder (The default location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Computing
Synonymous and Non-synonymous Distances Example 7.1: Activate the "HLA-3Seq.meg" file. If
necessary, refer to Example 1.2 in the “MEGA Basics” tutorial. From the main MEGA window launch bar, select Selection | Codon-based Z-Test of
Selection. An Analysis Preferences window appears. For
the Model/Method, select the Nei-Gojobori method (Proportion)
model. In the Test Hypothesis (HA: alternative) row,
select Positive Selection (HA: dN > dS)
from the pull-down menu. From the Scope row, select the Overall
Average option. For the Gaps/Missing Data Treatment option,
select Pairwise Deletion. Click on
"Compute" to accept the default values for the remaining options. A
progress indicator appears briefly, and then the computation results are
displayed in a results window in grid format. The column labeled
"Prob" contains the probability computed (must be <0.05 for
hypothesis rejection at 5% level). The column labeled "Stat" contains
the statistic used to compute the probability. The difference in synonymous and
non-synonymous substitutions should be significant at the 5% level. Close the Test of Positive Selection window. Managing Taxa with Groups
The “Crab_rRNA.meg” file, which is used in this
tutorial, can be found in the MEGA/Examples
folder (The
default location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Defining
and Editing Groups of Taxa In MEGA, you can partition
data into distinct groups and then evaluate distances within groups, distances
between groups, and the net distance between groups. Example 8.1: From the main MEGA window, activate the data present
in the "Crab_rRNA.meg" file. If necessary, refer to Example 1.2 in
the “MEGA Basics” tutorial. From the main MEGA window launch bar, select Data | Select Taxa and Groups.
Notice the left pane called Taxa/Groups and
the right pane labeled Ungrouped Taxa. Press the New Group button found below the Taxa/Groups
pane to add a new group to the data. Name this new group “Pagurus” and press Enter. While holding the Ctrl button on the
keyboard, click on all of the items in the Ungrouped Taxa pane that begin with Pagurus. This will highlight them. When
they are all highlighted, press the left-facing arrow button found on the vertical
toolbar between the two panels (make sure the Pagurus group on the left side is also highlighted otherwise the
arrow will not appear). Select the All group in the Taxa/Groups
panel and press the + (add) button found on the vertical toolbar between the two
window panes to add a second group. Name this group "Non-Pagurus". Add the remaining unassigned taxa to this group by
using the left arrow and press the Close button at the bottom of the
window to exit this view. Note: Now that groups have been defined, the Compute Within Group Mean,
Compute Between Group Means, and Compute Net Between
Group Means menu commands from the Distance option on
the launch bar may be used to analyze the data. Close all of the open windows. Computing Sequence Statistics
The “Drosophila_Adh.meg” data file, which is used in this tutorial, can
be found in the MEGA/Examples folder (The default
location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory).
The Sequence Data Explorer
provides various tools for visually analyzing sequence data as well as
calculating compositional statistics. In the following examples we will
demonstrate the basic usage of the Sequence
Data Explorer. Example 9.1: Activate the "Drosophila_Adh.meg" file). If
necessary, refer to Example 1.2 in the “MEGA Basics” tutorial. Select the Data
| Explore Active Data (F4) command. Use the arrow keys on your keyboard or the mouse to
move from site to site. At the bottom left corner of the window, you will find
an indicator that displays the column and the total number of sites. As you
move through the columns, the column indicator changes. Highlighting If you look at the bottom
of the Sequence Data Explorer window,
the Highlighted Sites indicator displays "None" because no
special site attributes are yet highlighted. You can highlight variable sites in various ways: ·
Select
the Highlight | Variable Sites
main menu option on the Sequence Data
Explorer main screen. ·
Click
the icon labeled V from the launch bar. ·
Press
the V key on the keyboard. Example 9.2: Use one of the above
methods to highlight variable sites in the Drosophila data. All sites that are
variable are now highlighted. The Highlighted indicator at the bottom of
the window has been replaced with the Variable
indicator. The number of sites which are variable is displayed, along with the
total number of sites (Variable sites/Total # of sites). When you press the V
key again, the sites return to the normal color. The Highlighted
indicator again displays "None". Now highlight the parsimony-informative sites by
pressing the P key, clicking
on the button labeled Pi from the shortcut bar below the main
menu, or selecting the Highlight |
Parsim-Info sites menu option. The Highlighted
indicator turns into the Parsim-info
indicator. To highlight 0,
2, and 4-fold degenerate sites, press the 0, 2, or 4 keys, respectively, or click
on the corresponding buttons from the shortcut bar below the main menu, or
select the corresponding command from the Highlight menu. Once
again, the Highlighter indicator will
turn into the Zero-fold indicator, Two-fold indicator, and Four-fold indicator respectively. Statistics The Statistics main
menu option allows you to calculate Nucleotide
Composition, Nucleotide Pair Frequencies
and Codon Usage. Before selecting one
of these options, you will need to select whether to use all sites or only the
highlighted sites. You will also need to select the format in which you want
the results displayed. Example 9.3: Select Statistics
| Use All Selected Sites. To
display the results of the calculation in a text file using the built-in text
editor, click the Statistics
menu option again and select the Display Results in Text Editor
option. To calculate the nucleotide base frequencies, select the option, Nucleotide
Composition, from the Statistics
menu. To compute codon usage, go back to the Sequence
Data Explorer and select the Statistics
| Codon Usage menu command. This will calculate the codon usage and
display the results of the calculation in a text file using the built-in text
editor. To compute nucleotide pair frequencies,
select the Statistics | Nucleotide
Pair Frequencies | Directional (16 pairs), or the Statistics |
Nucleotide Pair Frequencies | Undirectional (10 pairs) main menu
option.
This will calculate the pair frequencies and display the results of the
calculation in a text file using the built-in text editor. Note: Notice that
the Amino Acid Compositions option on
the Statistics menu is disabled
(grayed-out). This option is only available if the sequences have been
translated. Using the Amino Acid Composition Option Example 9.4: To translate these protein-coding sequences into
amino acid sequences and back again, select the Data | Translate Sequences main menu command from the Sequence Data Explorer window.
Once the sequences are translated, calculate the
amino acid composition by selecting the Statistics
| Amino Acid Composition main menu command from the Sequence
Data Explorer window. Close the Text
File Editor and Format Convertor window without saving your work. Close the
Sequence Data Explorer and select Close Data icon on the main MEGA
window. Building Trees from Distance Data
This tutorial illustrates procedures for building
phylogenetic trees using distance data. The “Hum_Dist.meg” data file, which is used in this
tutorial, can be found in the MEGA/Examples
folder (The
default location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Making a
Phylogenetic Tree from Distance Data Example 10.1: Activate the "Hum_Dist.meg" file. If
necessary, refer to Example 1.2 in the “MEGA Basics” tutorial. From the main MEGA window, select Phylogeny
| Construct/Test Neighbor-Joining Tree from the launch bar. The Analysis Preferences window will appear.
For distance data files, all of the options shown here cannot be changed. Click
on the button labeled Compute. A progress meter will
appear briefly. The Tree Explorer will display a
neighbor-joining (NJ) tree on the screen when the analysis completes. From the Tree Explorer launch bar, click on the i icon. The number of tabs shown here depends on the
type of tree that was constructed. For a Neighbor-Joining tree, the tabs are General,
Tree and Branch. Take a look at each to see the information they
contain. Saving your
Results MEGA allows you to save trees in MEGA’s native format or in the Newick
format. Example 10.2: From the Tree Explorer window, select File | Save Current
Session. In the Save As dialog, use the Save in drop-down menu to select the location, and then type in a
name for the session in the File Name
area. The tree will be saved with the MEGA ".mts" extension. Now, from the Tree Explorer window, select File | Export Current Tree from the main menu.
In the Save As dialog, use the Save
in drop-down to select the location. In the File Name area, type a name for the session. The tree will be saved
in Newick format with the ".nwk" extension. Go to the File
menu and click on the Exit Tree Explorer option. Constructing Likelihood Trees
MEGA
provides options for performing various calculations relating to likelihood. In
this tutorial, we will focus on the one you'll probably use most often,
constructing Maximum Likelihood trees. The “Drosophila_Adh.meg" data
file, which is used in this tutorial, can be found in the MEGA/Examples folder (The default location for Windows users
is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the user’s home directory). Constructing your Tree Example 11.1: Activate the "Drosophila_Adh.meg" file). If
necessary, refer to Example 1.2 of the “MEGA Basics” tutorial. Select Phylogeny
| Construct/Test Maximum Likelihood Tree option from the main MEGA
window launch bar. The Analysis Preferences window will appear.
For the Drosophila data file, you can choose between Nucleotide and Amino Acid
substitution types. Select Amino Acid. Now, click on the drop-down for Models/Methods. Note the
models available. Notice that the option to Select Codon Positions is
disabled for Amino Acid sequences. Change the Substitution Type to Nucleotide.
The list of Models/Methods changes, showing only models which are
applicable to nucleotide sequences. Select the Tamura-Nei model. Note that
the option to Select Codon Positions is now available. Click on the
button labeled Compute. A progress
indicator will appear briefly. The Tree Explorer will display the resulting
Maximum Likelihood tree on the screen. From the Tree Explorer toolbar, click on the i icon. The number of tabs shown here
depends on the type of tree that was constructed. For a Maximum Likelihood
tree, the tabs are General, Tree, Branch and Character
States. Take a look at each to see the information they contain. Saving your Tree MEGA allows you to save trees in MEGA’s native
format or in the Newick format. Example 11.2: From the Tree Explorer window, select File | Save Current Session from the main menu. In the Save As dialog, use the Save in drop-down to select the location
then type in a name for the session in the File
Name area. The tree will be saved with the MEGA ".mts" extension. From the Tree Explorer window, select File | Export Current Tree from the main menu. In the Save As dialog, use the Save in drop-down to select the location
then type in a name for the session in the File
Name area. The tree will be saved in Newick “.nwk” format. From the Tree Explorer window,
select File | Exit Tree Explorer from
the main menu. Click the Ok button without saving. Editing Data Files
There may be times when you want to make
changes to a data file. With the MEGA Alignment Explorer, you can
rearrange the taxa, delete blocks of taxa or delete blocks of sites. The
altered data file can then be saved in either MEGA or FASTA format. The “Chloroplast_Martin.meg"
data file, which is used in this
tutorial, can be found in the MEGA/Examples
folder (The default location for Windows users is C:\Program Files\MEGA\Examples. The default location for Mac users is $HOME/MEGA/Examples, where $HOME is the
user’s home directory). Using Alignment Explorer Example 12.1: From the main MEGA window, select Align
| Edit/Build Alignment. Select Create new alignment | DNA. Then click
Data
| Retrieve sequences from a file and press the Ok button. In the Open window, find and select the
"Chloroplast_Martin.meg" file. Rearranging Data Example 12.2: In the Alignment
Explorer window, click the row header for the row named Pinus. Hold the left mouse button down
and drag the row up, then release the mouse button when the position indicator
is just below the Porphyra row. Deleting rows Example 12.3: Now, click the mouse to highlight Porphyra. Select Edit | Delete on the main
menu of the Alignment Explorer. Do
the same for the row Pinus. Deleting sites Example 12.4: Click on the horizontal scroll bar at the bottom of
the Alignment Explorer window. Drag it all the way to the right. Now
click on any cell in the last column. Notice that the Site # display
changes to show the highest-numbered site, 11039. You can delete blocks of sites in the same way that
you can delete rows of data. Click on the gray header above any column of
sites, hold down the left mouse button and drag across to any other column
header to select multiple columns. On the toolbar, click the X
icon to delete the selected sites. Save the altered data file Example 12.5: On the Alignment Explorer menu, click on Data,
and then select Export Alignment. Choose either MEGA format, FASTA
format, or the PAUP format. In the Save As window, select the folder in
which you want to save your data file and then type a name in the File Name area. Click the Save
button. Close the Alignment Explorer and click Ok without saving. |
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