Analysis of Mitochondrial DNA Sequences Using BLAST, CLC, Clustal
and MacClade Analyses.
Introduction:
Evolutionary relationships between species are often diagrammed as trees. The
trees consist of a root that represents a single common ancestor for the whole tree,
internodes that represent either real or hypothetical common ancestors for specific
lineages within the tree, branches that describe relationships between ancestral species
and their descendents, and terminal nodes that represent the taxa being studied.
taxa
Terminal nodes
Internodes
Common ancestor
of Aliopo and
Tilonotila
Branch
Common ancestor of all
four taxa
DNA, RNA and protein data can all be used to generate such trees, (as can more
traditional characters, such as the presence or absence of a placenta). The assumption
that is made in the molecular analyses is that closely related species will have more
similar gene or protein sequences than distantly related species. Furthermore, since
mutations are both rare and random, it is assumed that the same mutation is unlikely to
arise in two different lineages. Thus, if you have four species with the following DNA
sequences: GAATTC, GATTTC, GATCTC, and GAGTTC, it would be more
parsimonious to arrange the tree as shown in “A”, where the AT mutation at the third
position only happens in one lineage, and the tree itself only requires three mutations,
than in “B”, where it there are four total mutations required to generate the tree, and the
AT mutation happens in two separate lineages.
GATTTC GATCTC
GAGTTC
TC
AG
GATCTC
AT
TC
GAGTTC
GATTTC
AG
AT
AT
GAATTC
Tree “A”
GAATTC
Tree “B”
Procedure:
BLAST Analysis of Human Mitochondrial DNA Sequences.
a) Download the Class Mitochondrial DNA Sequences as instructed by your
professor and find your number. If you are not there, your sequence was not of
sufficient quality to analyze. Do not worry, simply choose another sequence and
click on the link.
b) Select the entire sequence record, starting with > and ending with the last base
of the sequence. That format is called 'FASTA', and is a common sequence format
for almost all sequence analysis programs. Copy the sequence, and then go to the
NCBI BLAST database at http://blast.ncbi.nlm.nih.gov/Blast.cgi.
c) First, select nucleotide BLAST, as you did for your pre-lab activity, and paste
your sequence in the BLAST window. Is your sequence human mitochondrial
DNA?
d) Next, go back to the BLAST home page, and scroll down to the 'Align two
sequences (bl2seq)' link in the 'Special' window. Align your sequence with that of
Rarely Reclusive. Is it a perfect match?
Worksheet Question 1: Give the E value, the percent identity and the Score. Do
you think that Rarely is closely related to this you? Why or why not?
Phylogenetic Analysis - Creating Alignments and Trees Using UPGMA Analysis
During this part of the activity, you will learn how to use the CLC Free Workbench to
align DNA sequences and to generate a UPGMA tree.
Now you are ready to align your sequences so that you can perform your phylogenetic
analysis. Creating a sequence alignment makes sure that you are comparing homologous
portions (beginnings to beginnings, and ends to ends) of all of the genes in the Genetics
project.
a) Open up the CLC Free Workbench program from your dock. Import the class
FASTA sequences by clicking on the small import folder icon in the upper
toolbar.
b) In the Toolbox window (below the Navigation Area) double click on
'alignments and trees' and then on 'create alignment'. Click on 'class sequences'
and then click on the right arrow to move the sequences to the right analysis
window. Click on 'next'.
c) Under Gap settings, set Gap Open Cost to 100. This will discourage the
computer from trying to insert gaps in your sequences while matching them up.
d) Under End Gap Cost choose 'Free'. Since our sequences differ in length
because of the quality of your sequencing reactions (rather than a real
evolutionary difference) this setting will keep the computer from paying too much
attention to sequences that are longer or shorter than one another.
e) Make sure that 'Fast Alignment' is checked, and click on 'Finish'.
f) The computer will now align all of the sequences to each other (as opposed to
simply aligning two sequences as you did in BLAST (bl2seq). The aligned
sequences will open in a large window to the right of the Navigation Area. The
computer also generates a consensus sequence for all of the data, which represents
the most common sequence that all of the class sequences share. You can see the
consensus sequence below the aligned class sequences. If you scroll up and down
the alignment window, you can see that the matches are not perfect, and that the
computer has inserted many gaps to make the sequences align.
g) Now it is time to generate a phylogenetic tree from your aligned sequences.
Under the toolbox, double click on Create Tree. In the Selected Elements box you
should see 'mito124.txt_alignment'. Click on 'Next'. Under 'Algorithm' choose
UPGMA to tell the computer to use that method to construct the tree. Make sure
that the boostrap analysis is selected for at least 100 replicates. You can go up as
high as 1000 replicates.
h) Click on 'Finish'.
i) You can now see the UPGMA Phylogenetic Tree in your main window.
j) In the right window, under 'Tree Settings', choose 'Tree Layout' -> Node
symbol - > 'None' and make sure 'Layout' is set to 'Topology' (the branch lengths
will not be proportional to the UPGMA values as they would be if you choose
'Standard', but they will be easier to see).
k) Under 'Annotation Layout' make sure that 'Branches' are set to length, rather
than the bootstrap value.
Worksheet Question 2: Which group members are most closely related to one
another?
Worksheet Question 3: Which group members are most closely related to Rarely
Reclusive?
l) Finally, you need to export your tree in .jpg format for your worksheet. Make
sure your tree window is selected, and then click on 'Graphics' in the upper
program bar. Make sure you have chosen JPG formatting from the drop down list.
Make sure your file name has .jpg at the end. Choose to save the whole view in
high resolution (the middle value), then click on 'export'. Save the file to your
desktop.
You should see something like this, when you open the picture file:
Worksheet Question 4: Import your picture file into the appropriate space in the
worksheet.
Worksheet Question 5: Interpret this phylogenetic tree. Which one of your
group members is most closely related to Rarely Reclusive?
Worksheet Question 6: Which one of your classmates is most closely related to
Rarely Reclusive?
Worksheet Question 7: For each group member, state the person to whom they
are most closely related, according the UPGMA analysis.
Phylogenetic Analysis - Human Evolution and Your Place in the Human Family
a) Well, you can't all be most closely related to Rarely, so who are you related to?
You will now do Clustal Analysis to determine the answer to this question.
b) Follow this link (http://www.bioservers.org/bioserver/) to the Dolan DNA
Learning Center login page. Create a username, a password, and log in to the
Sequence Server site.
c) Click on 'Manage Groups' from the menu in the center of the top of the page.
You will now see a window that looks like this:
d) Click on the upper right pull-down menu (sequence sources). You should now
see a variety of choices. Choose 'modern human mt DNA'. Click on the boxes to
the left of each of the sequences to select them, and then click on 'ok'.
e) Click on 'Manage Groups' again. This time select 'Prehistoric Human mt DNA'
and click on all of the boxes, and then 'ok'.
f) You have now uploaded a whole bunch of mtDNA sequences from the database
for analysis.
g) To upload your own sequence, go 'Manage Groups' one last time. This time
select 'Public’ and click on ‘Susquehanna Genetics 2009’. Select your sequence,
and the sequences of the members of your group, and mitorarely (Rarely
Reclusive).
h) To compare your group members to the available sequences, click on the box
to the left of your sequences, and then next to any other sequences that interest
you (you'll see lots of drop-down possibilities within each group). You may only
select up to ten total sequences (including your group sequences). Have fun, but
also use sequences that make sense, given what you know about your ancestry.
i) After you have selected 10 sequences for analysis, find the word 'compare' in
the gray bar menu, and choose 'phylogenetic tree' from the drop-down box. Click
on "Compare". After your sequences are analyzed, a popup window will show
you your tree. This tree is based on an alignment program called “Clustal”.
Clustal Analysis generates a pairwise score for every pair of sequences that are to
be aligned, much like we did with the CLC alignment program. These scores were
calculated as the number of identities in the best alignment divided by the number
of residues compared (gap positions are excluded).
l) Choose 'phenogram' and 'yes' (to make the tree branch lengths proportional to
the evolutionary distances). You will see something that looks like the picture
below, but that contains the individuals and species that you chose to analyze.
m) Phenograms (phylograms) such as this one can provide a ton of information.
First of all, a phylogram is assumed to be an estimate of a phylogeny. IN other
words, the branch lengths are proportional to the amount of inferred evolutionary
change. A cladogram, on the other hand, is a branching diagram (tree) assumed to
be an estimate of a phylogeny where the branches are of equal length. Therefore,
cladograms show common ancestry, but do not indicate the amount of
evolutionary "time" separating taxa. One thing that this phylogram shows is that
Lake Mungo Man and African American #1 share a common ancestor, to which
Lake Mungo Man is more closely related.
Worksheet Question 8: Right click on the phenogram (command click on the
Mac) and select ‘copy picture.’ Paste your phenogram into the appropriate space
in the worksheet.
Worksheet Question 9: Interpret your phenogram. Which human ancestral
lineages is each of your group members most related to?
Worksheet Question 10: Which one of your group members is the most closely
related to Rarely Reclusive according to this Clustal-based analysis?
Worksheet Question 11: Does this analysis agree with your CLC-based
analysis?
MacClade Analysis of Mitochondrial Sequences and Maximum Parsimony
In this exercise you will use MacClade to generate cladograms or phylograms (you can
choose) that have the smallest tree length (this is analogous to the total number of
mutations needed to account for the tree) while minimizing the number of times that a
single mutation arises in multiple lineages. You will then compare your MacClade tree to
the ones you generated using the CLC/UPGMA and Clustal-based analyses.
Procedure:
Download the National Biomedical Research Foundation (NBRF) Format File from the
syllabus (filename mitochondrial.NBRF) to your desktop by typing control+click on the
file name.
Open MacClade
1. Double Click on the MacClade Icon.
2. Choose ‘Open File’ (Your computer may open a Finder Window
instead of allowing you to choose Open File..this simply takes you
directly to step 3, so proceed!).
3. Open ‘mitochondrial.NBRF’ from your desktop.
4. Click ‘OK’ to verify that your file is an NBRF DNA file.
5. You should now see that the mitochondrial sequence files have been
uploaded into the MacClade program as seen in the picture below:
6. While this window contains all of the sequences (the taxa in this case
are the individual students, and the characters are the individual DNA
bases at each position of the sequence), the sequences are not yet
aligned, so we need to do that right after you save your data.
Save Data
1. Under FILE choose SAVE FILE AS, and give your file a name and
save it to the desktop. This will make sure you do not lose your data if
something goes wrong during later manipulations. Don’t forget to throw
away all of your files and to empty the trash before you put your computer
away for the semester.
Aligning DNA Sequences
1. In order to analyze how closely related these gene sequences are using
MacClade, you need to make sure that each sequence is properly lined
up.
2. MacClade’s Alignment Tool can be seen in the toolbox in the bottom
left of your MacClade window, as indicated by the arrow below:
3. Click and hold on the Alignment Tool. In the popup box, select ‘slow
method using less memory’. If you do not do this, the program will
crash!!!
4. To align two sequences, make sure that the alignment tool is selected.
Now, click on the lower sequence, and drag it up to the one directly
above it. Release. The computer will now line up the two sequences
so that they match as closely as possible. Save your data. I
recommend you save after every alignment step below (sometimes this
program crashes)
5. Repeat by dragging the third sequence up to the second, and so on,
until you have finished aligning all of the sequences to the sequence
above them in a pairwise fashion (yes it’s a pain to only align them
pairwise…but that is all this program can do).
6. When you finish, you should be able to see (the bases are each colorcoded) that the sequences line up really nicely now. You are ready to
generate a phylogenetic tree.
Manipulate Data
1.
Generate tree
a) Under WINDOWS choose TREE WINDOW
b) Choose DEFAULT LADDER - a tree should now appear! This tree makes no
assumptions at all about how your microbes are related. In fact, if you look at the
tree, it simply places the organisms in the same order in which you entered the
sequences.
c) Under TRACE choose TRACE ALL CHANGES. This will provide a color
visualization of the number of nucleotide changes between a common ancestor
and the next ancestor or terminal taxon. If you place the cursor arrow on a tree
branch, the number of unambiguous nucleotide changes in that branch will be
written in the small box on the bottom right.
d) Under Display choose ‘Tree Shape and Size”. The choices, from left to right
below are either an angled or a square branch cladogram, or a phylogram.
2.
Minimize Tree
a) Find the most parsimonious tree by clicking on branches, and then dragging
them to new tree locations, remembering as you do so that you are changing the
assumptions about how the species are related. Your goal is to search for the tree
that gives the lowest number of “steps” (analogous to the total number of
nucleotide changes necessary to account for the proposed evolutionary
relationships - so lower is better). If you want to know what the theoretical
smallest tree is, you can choose    minimum possible from the upper
program bar. Both the current tree length and the minimum possible tree length
will be displayed in a small box in the lower right hand corner of your tree. Try
to get as close to the minimum as possible.
b) Try manipulating the tree so it looks like the ‘ideal’ tree from your Dolan DNA
Center analysis. Have you found the minimum yet?
c) Now that you’ve played around with the tree a bit, it is time to tell you a little
secret – the computer will actually help find the smallest tree for you. Simply
click on the ‘search above’ icon in the toolbox, and then move the cursor to the
root of the tree. When you click on the root, the program will search for, and
display, the shortest tree above that spot.
d) Once you are convinced you have the smallest tree, save your file and then go
to File>Save Graphics File. Choose either a phylogram or a cladogram from the
left menu, and then under Options in the right drop down menu choose Legends
and click on ‘trace legend’ and ‘tree statistics’. Under Options “Branch Shading”
choose colors. Click on PICT File and save the file to your desktop.
Worksheet Question 12: Import the PICT file into the appropriate spot in the
worksheet.
Worksheet Question 13: Interpret this phylogenetic tree. Which one of your
group members is most closely related to Rarely Reclusive?
Worksheet Question 14: Which one of your classmates is most closely related to
Rarely Reclusive?
Worksheet Question 15: For each group member, state the person to whom they
are most closely related, according to the MacClade analysis.
Worksheet Question 16: Does this analysis give you a different answer than the
other two analyses did? If so, why do you think that is?
Workshop Genetics Worksheet: Analysis of Mitochondrial DNA Sequences Using
BLAST, CLC, Clustal and MacClade Analyses.
Names__________________________________________________________________
Worksheet Question 1: Give the E value, the percent identity and the Score. Do you
think that Rarely is closely related to this you? Why or why not?
Worksheet Question 2: Which group members are most closely related to one another?
Worksheet Question 3: Which group members are most closely related to Rarely
Reclusive?
Worksheet Question 4: Past your picture file here.
Worksheet Question 5: Interpret this phylogenetic tree. Which one of your group
members is most closely related to Rarely Reclusive?
Worksheet Question 6: Which one of your classmates is most closely related to Rarely
Reclusive?
Worksheet Question 7: For each group member, state the classmate to whom they are
most closely related, according the UPGMA analysis.
Worksheet Question 8: Paste your phenogram here.
Worksheet Question 9: Interpret your phenogram. Which human ancestral lineages is
each of your group members most related to?
Worksheet Question 10: Which one of your group members is the most closely related
to Rarely Reclusive according to this Clustal-based analysis?
Worksheet Question 11: Does this analysis agree with your CLC-based analysis?
Worksheet Question 12: Import the PICT file here.
Worksheet Question 13: Interpret this phylogenetic tree. Which one of your group
members is most closely related to Rarely Reclusive?
Worksheet Question 14: Which one of your classmates is most closely related to Rarely
Reclusive?
Worksheet Question 15: For each group member, state the person to whom they are
most closely related, according the MacClade analysis.
Worksheet Question 16: Does this analysis give you a different answer than the other
two analyses did? If so, why do you think that is?