Bioinformatics and Phylogenetics of Fungi

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(Written by Benjam
in Wolfe and Primrose Boynton.
If you have questions, email Anne
Pringle at pringle@oeb.harvard.edu)


OEB 54


Lab 10

Bioinformatics and Phylogenetics of Fungi

November 10, 2009


As part of her research, Primrose has isolated and sequence
d DNA from yeasts from pitcher
plants in the wild. In this lab exercise, you will use bioinformatics databases to determine the
groups of fungi that
some of
these sequences belong to. You will also create a phylogenetic
tree to look at a finer scale how t
hese environmental isolates are related to known fungal
species.

IDENTIFYING ENVIRONMENTAL FUNGI:

Imagine you are Primrose, and have some water containing unknown types of fungi. How
can you identify the fungi? There are two scenarios, and we’ve already

encountered one.

Culturable fungi:

Write down a schematic of the procedure mycologists use to identify culturable fungi here.






Unculturable fungi:

Many fungi can’t be cultured. Here’s a schematic of a procedure mycologists use to identify
unculturable

fungi.

1.

sample collection


DNA extraction


PCR with fungal
-
specific primers





2.

clone PCR products (if you have a mixture)


sequence clones


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What do you do with a sequence?

BLAST SEARCHING:

A DNA sequence is just a series of As, Gs, Ts, and Cs. What does this data mean, and how
do we use it? Today, we will look at sequences from Primrose’s project. Her primers
amplified parts of the ITS (internal transcribed spacer) region
. There are two types of rDNA
transcripts: subunits and spacers. Subunits go on to become parts of ribosomes, while
spacers don’t. As a result, spacers tend to evolve more quickly than subunits.

1.

Download sequence data and software from the “
Bioinformatics
and Phylogeny
Lab
” folder
on the course website
.



2.

Download and open the “pp_yeast_for_BLAST.fasta” file. There are three
sequences named pp_yeast_1, pp_yeast_2, and pp_yeast_3.


3.

Using a web browser, navigate to the NCBI BLAST website:

a.

http://blast.ncbi.nlm.nih.gov/Blast.cgi


b.

Click on the “nucleotide blast” link under Basic BLAST
.


4.

Copy and paste one of the three sequences into the BLAST search area (into the
white box where it says “Enter accession

number, gi, or FASTA sequence”). Be sure
that in
Choose Search Set
, “Others” is chosen and for
Program Selection
,
“Somewhat similar sequences (blastn)” is chosen
.


5.

Click “BLAST” to perform your search.


6.

Results should appear in a few seconds.
What funga
l species names come up in
the list of BLAST matches? Through Wikipedia or Google searching, what
can you learn about the natural history of these environmental sequences?





VIEW ALIGNED SEQEUENCE DATA:


1.

Download the sequence alignment “yeast_its.nex” t
o view a DNA
sequence alignment of pp_yeast_3 and closely related fungi. Open this
file.


2.

The sequence data from each sample or taxon is displayed in a separate row.
The sequences have been aligned, so that regions of the DNA that have identical
nucleotid
es between taxa line up in columns


3.

Inspect…

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i.

How similar are the sequences?

ii.

How would you group the taxa into those that are most similar?

iii.

Are there regions of the DNA that are more and regions that are
less similar?

iv.

Is there one sequence that is more diff
erent from the others?





MAKE A PHYLOGENETIC TREE:


1.

Make sure the file “yeast_its.nex” is saved to the desktop.


2.

Go to Spotlight in the upper right hand corner of the screen and type
GARLI
. This
will locate the program GARLI. Open the program. Not muc
h will happen.
(GARLI is free and can be downloaded at
http://www.bio.utexas.edu/faculty/antisense/garli/Garli.html
)


3.

Go to FILE > Open.. and locate
“yeast_its.nex”

on the desktop (where you just
saved it).


4.

Listen to Primrose as she explains various feat
ures of GARLI. When done, click on
Run
.

The program will take 5 minutes or so to create a tree from the sequences. A
random tree is generated first, then new trees are created and compared to
the original tree for 10,000 generations. You can see the pro
gram list the
current generation, and the generation where the best tree so far was created


5.

When GARLI is finished running, a file will have been created on the desktop called

yeast_its.best.tre.” Open the application FigTree (using spotlight if necessa
ry), and
open this file.

(
FigTree is a free phylogenetic tree viewing program from
http://tree.bio.ed.ac.uk/software/figtree/

)


6.

In order to better visualize the relationships between t
he fungi, the tree should be
rooted to an outgroup fungus.

i.

Click on the long
Candida glabrata

branch

ii.

Click the yellow arrow “Reroot” button


Note where in the
phylogenetics tree

the pitcher plant yeast falls.


What
known
species is
this unknown fungus

cl
osely related to?





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