BLASTing Flu Viruses

amyC · Post by **amyC** » Tue Sep 15, 2009 8:17 am

Hi Swimmy - I have asked the author of the project to take a look at your questions from September 4 as you worked through the revised project. As soon as I hear back, I'll post his response here.

In the meantime, you might want to look back at the Science Buddies' information on "variables" to see if you can identify the variables in the project:
https://www.sciencebuddies.org/science- ... bles.shtml

Amy
Science Buddies

amyC · Post by **amyC** » Tue Sep 15, 2009 1:20 pm

Hi Swimmy -

I've got some answers for you.

You asked:

"This is about the H1N1 virus. It says the virus is related to a part of the vaccine for seasonly flu.
Should I use those strains mentioned as the vaccines? "

Answer: Yes, I would use the A/Brisbane/59/2007 sequence since that is a component of the 2008-9 vaccine. The protein sequence can be found here: http://www.ncbi.nlm.nih.gov/protein/ACA ... e_RVDocSum.

Click on "BLAST Sequence" on this page. A new page appears.

Click on "BLAST" on the next page to BLAST the protein against a protein database. The default database is the non-redundant database of proteins at NCBI, which includes almost every protein sequence that has been published. The default number of alignments is 100. You can click on "Algorithm parameters" to increase the number of alignments. You should spend some time looking up more about the BLAST tool using the links provided on the BLAST page, for example http://blast.ncbi.nlm.nih.gov/Blast.cgi ... =BlastDocs.

To limit the database to sequences that were found in a particular year, for example 2009, type "2009 [WORD]" in the box that says Entrez query on the BLAST page. This might be useful to look at changes over time.

The output includes identification of a "Putative Conserved Domain". The "Putative Conserved Domain" output tells you what protein family your input sequence belongs in. This is found by comparing your input sequence to a database of conserved sequences for various kinds of proteins. This is information that is included in addition to the BLAST output. BLAST just identifies similar sequences and aligns them. The input belongs in the hemagglutinin family, which makes sense since you are using the HA protein (influenza hemagglutinin) as an input. If you click on the image of the hemagglutinin you can learn more about this family of proteins. If you want to learn more about how this tool works, see this paper: http://www.pubmedcentral.nih.gov/articl ... tid=308855.

Scroll down to see the BLAST output. There are 20 amino acids in proteins. The BLAST output uses the standard 1 letter code to identify amino acids. You can learn about these here: http://www.biology.arizona.edu/biochemi ... aa/aa.html. You can find similar info on other sites on the web or a good biology textbook.

You asked:

I tried the method in the revised edition of BLASTIng Flu Viruses. I am successful with the procedure but then it says "Putative conserved domains have been detected, click on the image below for detailed results." What does this mean?

Answer: See explanation above.

Also, the results are kind of confusing, because I don't see the alignments...

Answer: See explanation above regarding how to get BLAST alignments.

You said:

also, there are no lowercase letters. I'm not really sure if what I am seeing is correct, if it is correct, I don't know how to analyze the results.

Answer: Lower case letters are used in BLAST outputs to show where sequence with low complexity, such as a sequence like AAAAAAAAAAAAA, were filtered out. These low-complexity sequences are not very informative and confuse the BLAST output interpretation. There are none in the HA sequence

You asked:

What do the plus signs (+) and the spaces mean?

Answer: Plus signs mean that the amino acid change is considered "conservative". In other words, it is a change that is often found in other proteins and probably won't disrupt the structure of the protein.

You asked about your hypothesis:

If I had the title "Which regions of the influenza virus protein are most subject to change?", what could I write for my hypothesis?

This is up to you. Might be something like "not all regions will change equally because …."

Do I just specify the kind of protein?

Yes

Would I need to cite where I got the protein sequence?

Yes

would i need to cite the websites in the procedure?

Yes

He also notes that standard controlled, dependent, and independent variables might not apply in the traditional way for the type of project you are doing.

I hope this helps!

Amy
Science Buddies

swimmy · Post by **swimmy** » Tue Sep 15, 2009 6:07 pm

Thanks so much.
I understand the different types of variables, but in this project there isn't really anything that was changed.
Wait, so if there are no lowercase letters in HA, then how could I tell which region of protein has more chance to mutate?
And how do I define the region of protein, by the types or where they are located in influenza? But how could I tell where they are located by the BLAST search?

Thanks,
swimmy

amyC · Post by **amyC** » Wed Sep 16, 2009 11:02 am

Hi Swimmy - I have answers for you once again.

I understand the different types of variables, but in this project there isn't really anything that was changed.

In a bioinformatics project such as Blasting the Flu, you are asking questions about differences (changes) in protein sequence. You will have to ask very focused questions or the blast output will be overwhelming.

Wait, so if there are no lowercase letters in HA, then how could I tell which region of protein has more chance to mutate?

By looking at the Blast alignments, you can see regions of the protein that are invariant, that is they never change, and regions that do vary. Regions that vary have blank spaces or +'s in the middle row of letters between the two sequences, indicating that the alignment breaks down.

And how do I define the region of protein, by the types or where they are located in influenza?

Define them by where they are located (their position by number) in the protein. If you study the protein structure (recommended) you can learn more about the function and structure of different protein domains.

But how could I tell where they are located by the BLAST search?

You can see the numbers for the amino acids in the Blast output. You may need to count from the ends. See the example below.

Query 121 YASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKYKYPAL 180
YASLRSLVASSGTLEFNNESF+WTGVTQNGTSSACKRRSN SFFSRLNWLTHLKYKYPAL
Sbjct 121 YASLRSLVASSGTLEFNNESFDWTGVTQNGTSSACKRRSNKSFFSRLNWLTHLKYKYPAL 180

There are changes at 142 and 161. The change at 141 is considered more conservative so a + sign is used.

If you are printing out alignments, use a font that keeps every letter the same width so the letters line up. Courier is a font that is often used.

As mentioned previously in a different post, you might want to pick a single mutation for your project to keep it focused. Then you could ask questions about how often this mutation is seen (% of all HA sequences), how does its prevalence change by year, what year did it first appear in the database, etc. You can use the mutated version of the sequence as the input in BLAST to find identical mutant sequences. You could also (more ambitious) try to see if the mutation appears independently in different populations or occurred just once and then spread.

Amy
Science Buddies

swimmy · Post by **swimmy** » Wed Sep 16, 2009 2:07 pm

Thanks. I will try this as soon as possible.

swimmy

swimmy · Post by **swimmy** » Wed Sep 16, 2009 5:27 pm

so for variables, would I need to change something in the BLAST input?

thanks,
swimmy

amyC · Post by **amyC** » Thu Sep 17, 2009 10:30 am

Hi Swimmy - I have some more information from one of our staff scientists about the "variables" in this project. I want you to try and think through the issue of identifying these.

The independent variable is the one that is changed by the scientist.

In this project, can you see what "you" selected to use with BLAST?

The dependent variable responds to changes made to the independent variable.

In this project, the dependent variable relates to the alignment.

Controlled variables are quantities that a scientist wants to remain constant.

What are you keeping the "same" for each of your searches?

See if you can use this information to come up with what the variables might be.

Amy
Science Buddies

swimmy · Post by **swimmy** » Thu Sep 17, 2009 3:53 pm

Independent: type of flu
Dependent: region where the protein mutates
Constant: Same type of BLAST search, same type of flu

Am I on the right track?
i want to compare which region of protein mutates in flu each year... or different strains. If I do each year, can I just do the most common flu that year?

swimmy · Post by **swimmy** » Fri Sep 18, 2009 10:03 pm

ok, i don't really get how you can see which number protein the mutation is. Is it 1-20 or the numbers such as 141?
also, what does more conservative mean?
what do you mean by one mutation?

swimmy · Post by **swimmy** » Sat Sep 19, 2009 2:21 pm

how would I tell where the mutation is with so many alignments?
do i just see where a mutation occurs in one alignment?
i have to turn in science fair application by tuesday, september 22.
I have to write my procedure, hypothesis, variables, make a chart/graph/data table and abstract by then.
If I compare different types of flu would I be able to make a pie chart?

swimmy · Post by **swimmy** » Sat Sep 19, 2009 4:15 pm

This is how the science fair application is like and how i think im going to enter it:

Student Participants: (I will not put my name in the forum)

Title of Project: Do flu viruses from different years change at the same regions of protein?

List your procedures/steps you'll complete:*
1) Go to the Flu Activity & Surveillance page at The U.S. Centers for Disease Control and Prevention (CDC) website: http://www.cdc.gov/flu/weekly/fluactivity.htm.

2) Click on "Go" next to the year 2008-2009 “Current Weekly Influenza Report”.

3) Read the information on the page. In particular, find the paragraph with the heading "Antigenic Characterization." It will tell you the name of the virus strains that are candidates as vaccines.

4) You can obtain the sequences for these strains from the NCBI GenBank website: http://www.ncbi.nlm.nih.gov/Genbank/.

5) Select "Protein" with the “Search” drop-down menu. Type in the name of the flu strain (use A/California/07/2009 (H1N1) in the search box.

6) Full-length HA is 566 amino acids. In order to retrieve full length-entries, add this text to the search box AND 566[SLEN]. Click "Go."

7) Click on the active link for the HA protein page.

Copy the accession number for the full-length HA protein. Click on the "BLAST Sequence" link (column on right side of page).

9) The BLAST page will pop up. The database should be set automatically to "non-redundant protein" and the algorithm should be "blastp." Click the BLAST button.

10) The BLAST report contains alignments of your search sequence against sequences in the database. The regions of the protein that is most likely to change show up as lowercase letters in the alignment.

11) Repeat Steps 1-10 with past flu seasons (2000-2008) and compare.

List the materials you will use: computer with high speed internet

Independent Variables: type of flu

Dependent Variables: region where protein is likely to mutate

Controlled Variables: same kind of BLAST search

Hypothesis:** My hypothesis is that viruses from different flu seasons do not mutate in the same regions of protein.

What research will you do before experimenting?:*** I will research BLAST (internet tool that I will be using), flu virus, flu vaccine, and the seasons of flu.

How will you display your results? (Create a table/graph/chart):****

*Please give me feedback for my hypothesis.
**In the application, I don't need to explain my hypothesis (as I was not supposed to conduct any experiment. I wanted to try this in case there was a problem...)
***Whoops... I did my research already. Have any suggestions for any other research?
****What should I make a table/graph/chart for? All I can think of is a pie chart comparing the regions of protein where the flu virus is most likely to mutate.

swimmy · Post by **swimmy** » Sat Sep 19, 2009 7:43 pm

Ok, I don't really get how I can get one mutation from all those strains of flu?

swimmy · Post by **swimmy** » Sat Sep 19, 2009 8:37 pm

If I compared mutations in types of flu by year, would it make more sense to limit it to only 2009 mutations (if I'm doing 2009) or just BLAST without specifying?

swimmy · Post by **swimmy** » Sat Sep 19, 2009 9:19 pm

The way I see it, if I do the year specified mutations, i get mutations for the flu that year, and if i don't i have to do the different types of strains.
Is this right?
I'm going to do it like this.

swimmy · Post by **swimmy** » Sat Sep 19, 2009 9:50 pm

Ok, so when I did the swine flu BLAST without specifying the year of mutations, I got the following:
Mutations by strain
H1N1 (2009)
-212 (4)
-101 (74)
-217
-333 (73)
-410 (2)
-443 (2)
-385
-166
-520
-8 (2)
-544 (2)
-467 (2)
-240 (4)
-225 (23)
-48 (3)
-476 (4)
-286 (4)
-75

The most common mutation is at 101/566

Is this how I would say the results?

And did I do the right thing by looking at the whole blast search for the mutations?

swimmy · Post by **swimmy** » Sat Sep 19, 2009 10:01 pm

Sorry if about my past post editing.
I don't have any questions about the stuff on the 1st page, so please just answer the questions on the 2nd page.

swimmy · Post by **swimmy** » Sun Sep 20, 2009 12:39 pm

If I compared the regions where flu from different year/strain mutate, what would be the motive behind it?
Would it give scientists an idea of how to make a vaccine?

MichaelD · Post by **MichaelD** » Mon Sep 21, 2009 9:44 am

swimmy wrote:If I compared the regions where flu from different year/strain mutate, what would be the motive behind it?
Would it give scientists an idea of how to make a vaccine?

I'm joining the conversation late so I apologize if any of this is has already been discussed. A retrospective analysis of genetic differences between strains is useful in looking at the evolutionary path of the virus. I believe that when new strains emerge, the genetic differences between them help scientists to select the appropriate epitope for vaccine generation.

Mike

amyC · Post by **amyC** » Mon Sep 21, 2009 12:10 pm

MichaelD - Thank you so much for joining the conversation. If you have comments on Swimmy's proposed application, I am sure they would be appreciated.

https://www.sciencebuddies.org/science- ... =25&t=5115

Swimmy, I have also passed your other questions to the Science Buddies' scientist who has been helping you. If MichaelD or another expert can step in and help, great!

Amy
Science Buddies

MichaelD · Post by **MichaelD** » Mon Sep 21, 2009 3:19 pm

Hi Swimmy,

I have perused this thread and see that a lot of progress has been made. If you list your outstanding questions and/or issues. I would be happy to try and get you through them.

Mike

swimmy · Post by **swimmy** » Mon Sep 21, 2009 3:53 pm

If you could make suggestions for the application (variables and graph), that would be great.

MichaelD · Post by **MichaelD** » Tue Sep 22, 2009 8:21 am

swimmy wrote:If you could make suggestions for the application (variables and graph), that would be great.

Hi swimmy,

I think for this type of study, you could pick one flu protein and track how it mutates year to year. One graph might plot number of mutations versus the different flu proteins, or you could concentrate on one protein and and plot number of mutations versus amino acid position in that protein. I think by doing this, you might find that one protein (or a handful of amino acid position in one protein) may be more susceptible to mutation than others. You could then go back and look at the significance of these proteins to the virus life-cycle.

Mike

swimmy · Post by **swimmy** » Wed Sep 23, 2009 7:51 pm

Thanks.
I can't change my topic anymore because I turned in my application and my science teacher accepted it...
So right now my question is, "Did the flu viruses from past flu seasons mutate at the same regions of protein"
where I compare where mutating regions of proteins were (by the most common mutation)

swimmy · Post by **swimmy** » Fri Sep 25, 2009 8:43 pm

I don't understand how to cite the sequences. Is it on the page where I get the hemagglutinin sequences?

swimmy · Post by **swimmy** » Sun Sep 27, 2009 5:21 pm

My teacher thought the reason why I am doing my project (Did flu viruses from different seasons mutate in the same regions of protein) was too vague.
She said to include what the significance is if the answer is yes and if its no.
I need help on this.
And is there a way to narrow my question down? Please explain.

swimmy · Post by **swimmy** » Sun Sep 27, 2009 9:07 pm

My teacher said I could narrow my topic down if I wanted as long as the procedure stays pretty much the same (using BLAST and interpreting the search)
And I still don't really get how to do the graph...

MichaelD · Post by **MichaelD** » Mon Sep 28, 2009 3:35 pm

swimmy wrote:My teacher thought the reason why I am doing my project (Did flu viruses from different seasons mutate in the same regions of protein) was too vague.
She said to include what the significance is if the answer is yes and if its no.
I need help on this.
And is there a way to narrow my question down? Please explain.

Hi swimmy,

I think there are there are two possibilities here:

1) Did flu virus from different seasons show mutations in the same proteins?
or
2) Did flu virus from the different seasons show mutations in the same region of the same protein?

Depending on which one of these you are concentrating on, the conclusions drawn will be different. If you find that a single protein is mutating between strains, then you can conclude that this protein has a critical roll in viral infectivity (and circumventing the latest vaccine). If you find that a set of proteins is simultaneously mutating between strains, then the conclusion will not be as clear.

In terms of graphs, you could plot frequency of mutation versus flu protein or frequency of mutation version amino acid position in a specific protein and compare across strains.

Mike

swimmy · Post by **swimmy** » Fri Oct 02, 2009 8:57 pm

I am doing "Did the flu virus from different seasons show mutations in the same proteins"
So for the reason would I just say that the protein is important in viral infectivity and working around the vaccine?
Also, what type of graph should I use? I'm thinking of a line graph.

swimmy · Post by **swimmy** » Sat Oct 03, 2009 2:12 pm

How about this?
What is the objective of this experiment?
The objective of this experiment is to see whether flu viruses from different seasons mutate at the same regions of protein. If a single protein is mutating between strains, then the protein can make the virus more contagious and therefore possibly more resistant to vaccines. If a set of proteins is simultaneously mutating between strains, then the information gathered will not be as clear.

swimmy · Post by **swimmy** » Sat Oct 03, 2009 2:14 pm

I would like to include "swine flu" or "h1n1" in my title.
How would I do this?

BLASTing Flu Viruses

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