Bioinformatics Assignment

fabulousgalaxyΒιοτεχνολογία

1 Οκτ 2013 (πριν από 3 χρόνια και 11 μήνες)

71 εμφανίσεις

Bioinformatics Assignment

(
BLUE

is the same assignment done on the Biology Workbench)


1. Translation of DNA into Amino Acid sequence


Log
on to the ISU Bioinformatics Portal (contact Mike Thomas, mthomas@isu.edu)


Username: #####


Password: #####


On the

left side you will see a listing of nearly 20 software programs. Click on the file labeled
EMBOSS
, click on
Nucleic
, click on
Translation
, click on
transeq


Below
input Section
, enter your DNA sequence in the box marked
Actual data


In the
advanced S
ect
ion
, under
Frame(s) to translate
, choose
All six frames


Scroll back up to
the top, change the default email address to your own (otherwise I will delete
any emails sent to me)


To begin the
search
, click on
Submit transeq

at the top of the page


When your

results are returned, you will see at the top of the page

Results:

click on
outseq.out


You will see 6 different results, one for each reading frame. If any sequences have an asterisk
(*), that means there is a stop codon. These sequences
with an *
are
meaningless and you should
immediately disregard that as an option. Copy the remaining sequences and proceed to the next
step.


1. Translation of DNA into Amino Acid sequence


Go to workbench.sdsc.edu, and register/log in.

Click on Session Tools, and the
n select “Start New Session”.

Give your session a name such as “Unknown Id” and click on the “Start New Session”
button.

Click on Nucleic Tools, and then select “Add New Nucleic Sequence” and “Run”.

Put a description of your sequence (such as Unknown Seq
uence 1) into the “Label:” box.

Copy and paste your sequence into the “Sequence:” box.

Scroll down the page and click on “Save” you will end up back in the Nucleic Tools
window.

Select your sequence by clicking in the check box.

Scroll to the bottom of the

available tools and select “SIXFRAME” and the “Run”.

You will be taken to the options page, click on the “Submit” button. The next screen shows
you the amino acid translations of your nucleotide sequence in all six reading frames.
Select the sequences tha
t have no *, which indicate stop codons, by clicking on the
check box. Then click on “Import Sequence(s)” at the bottom of the page.

Your translated sequence(s) are now in the Protein Tools section of the Workbench.


2. BLAST Protein search


Log on to the

following web address:


http://www.ncbi.nlm.nih.gov/BLAST/


Under
Protein
, click on
Protein
-
protein BLAST (blastp)


Enter your amino acid sequence in the box labeled
Search


Click on
BLAST!
, on the next
screen click on
Format!


This may take a few seconds to search all the available databases, so please wait patiently


Your file will be returned with a match to known amino acid sequences.

As you scroll down the screen you will see

Distribution of
Blast Hits on the Query Sequence
, keep scrolling down until you find

Related Structures

Sequences producing significant alignments:


Each sequence has a score (bits) , which tells you

how closel
y aligned the 2 sequences are. By
clicking on the
Score
, it will take you down to a match.

Each match

appears as follows:


Query: 1



Sbjct: 300
(or some other number)


Query

is the sequence you submitted for analysis.
Sbjct

is t
he match that was found. The middle
line is the computer’s attempt to align them for you.


Find 10 different species
(if available, use human as one species)
that share similarity to the
submitted sequence. Copy and paste this
Sbjct

sequen
ce into a new f
ile to be used for

phylogenetic analysis.

Each species must have a ONE word name, if the name consists of more
than one word, please use a one word abbreviation of 8 characters or less. Do NOT use the Latin
name of genus and species, find the common name.

The format must be as follows for this
sequence to be used in the next step:

>species name(return)

Amino acid sequence(return)

>species name(return)

Amino acid sequence(return)


>human

ANSNCVMFKLGIRKMRL

>frog

ANSDHYMKLGIKMRL


Write all 10 sequences like
this!!



2. BLAST protein search


Select one of your translated sequences by clicking on the check box.

Scroll through the list of tools, and select “BLASTP”, then click on “Run”.

From the list of databases, select “Genpept Full Release” and “Genpept Updat
es”. Do not
change any other options on this page.

Scroll down the page and click on “Submit”.

In less than a minute, your results will appear on the screen.
As you scroll down the screen
you will see “Sequences producing significant alignments:” Each sequ
ence has a score
(bits) , which tells you how closely aligned the 2 sequences are. By clicking on the
Score, it will take you down to a match. Each match appears as follows:


Query: 1



Sbjct: 300 (or some other number)


Query is the sequence

you submitted for analysis. Sbjct is the match that was found. The
middle line is the computer’s attempt to align them for you.


If the Expect value associated with the first Sbjct is not close to 0, then the reading frame is not
correct, and you will n
eed to repeat the above steps with your other translated sequences.


Find 10 different species (if available, use human as one species) that share similarity to the
submitted sequence. Scroll through the table at the top of the screen, and click on the
c
heck box for each sequence that you wish to select. Then click on the “Import
Sequences” button.

Select the ten sequences from the BLAST search by clicking on their check boxes.

Select “Edit Protein Sequence(s)” and click on “Run”.

Replace the Genpept:##
# that follows the > in the “Sequence:” box with the common name
of the species from which it comes. Each species must have a ONE word name, if the
name consists of more than one word, please use a one word abbreviation of 8
characters or less. Do NOT use

the Latin name of genus and species, find the common
name. Be careful to keep the > symbol exactly where it is.

When you have changed all of the Sequence files, click on the “Save” button, which can be
found at either the top or the bottom of the screen.





3. Phylogenetic Analysis


Back on the ISU Bioinformatics Portal, on the left side, click on
Clustalw
, click on
clustalw


Under Number 2.
Actual data
, paste in your 10 sequences in the format above


In the next box labeled
Actions
, use the drop down
menu to choose

-
tree: calculate NJ tree


Scroll down, under
Multiple Alignments Parameters


The second drop down box asks you to choose between Protein or DNA (
-
type), choose
protein


Scroll back up to the top of the page
, change the default Email to your
own,

and click on
submit
clustalw


If you did everything correctly, you will see under
Results:

a file named
infile.ph


Below this file is a drop down box, choose
drawgram


Click on the box next to this which reads
Run the selected program on infile.ph


A
new screen will pop up, under
Drawgram options


Choose the Tree style named
P: Phenogram


Scroll down to

Drawgram Options


Next to the box labeled,
Which plotter or printer will the tree be drawn on


Choose
P: PCX file format


Scroll back to the top of the

page and click on
Run drawgram


Your results will appear in a file named
plotfile.
pcx


Save this file to your computer.


2. Phylogenetic Analysis


Select your newly edited sequences, which are now labeled “edited”.

Scroll through the Tools, and select “CL
USTALW”, then click on “Run”.

Do not change the options, click on “Submit”

In less than a minute, you will see the aligned sequences. These sequences can be copied
and pasted into a word document, although you will lose the color. Click on the
“Import Alig
nment” button.

Your aligned sequences are now in the Alignment Tools section. Select your alignment,
scroll to and select “DRAWGRAM” and click on “Run”.

On the options page change “Exclude positions with gaps:” from no to yes and “Correct for
multiple sub
stitutions:” from no to yes. Click on the submit button.

You can download a postscript version of your tree. Click on the “Return” button when
you are done.



Do NOT print your assignment; send it to me via email. Please include the following
informatio
n in your assignment:

1.

The name of your protein

2.

An alignment of each of 10 different species with differences shown in
bold

or a different color (each sequence is compared only to the human not to each
other)

3.

A phylogenetic tree showing the evolutionary rel
atedness of each species