hello this is Bob Lessick at the Center for Biotechnology Education at Johns Hopkins University
in this exercise we will be examining the blast tool
blast is the basic local alignment search tool
it allows you to take a sequence and search a large database to find sequences that are related
to the sequence that you input
our objectives for today's exercise are to
pick the right blast program because there are several to choose from
once you've chosen the right blast program you'll want to enter the query sequence. Blast requires
a sequence to start with. Either a DNA sequence
or a protein sequence
from there we'll pick the appropriate database and there are usually a few choices there
then we'll actually run the blast search, look at the output and then analyze the output based on e-values
our starting point is the NCBI webpage which is
www.ncbi.nlm.nih.gov
one of the more used websites in bioinformatics
I'm going to click on the right menu
the link says blast
or I also go to that web address and after the nih address
just after the slash, just type in blast that would get you there as well
there are five blast programs
there is nucleaotide blast, protein blast and then there are three translated blast searches
which are a little bit more advanced
in general if you're going to use, if you're starting with a DNA sequence you would want to use nucleotide blast
if you're starting with a protein sequence, you would want to use protein blast
so I am going to open up
the sequence that we are going to use for this exercise
and the sequence is called rat example protein you can see from the letter code that this
is a protein sequence. These are amino acids not just the four nucleotides
and this sequences is in fast a format meaning that it's got a title plus the sequence
so I can put this on the clipboard right now
and copy
and because this is a protein sequence I'm going to open up protein blast
okay now here is the blast input page
and I can switch blast programs using these tabs at the top menu
Enter Query Sequences is where you are going to want to take your starting sequence
which is our rat sequence, so I am going to paste that in
so the sequences is in, including the title
and that will help identify the protein
and now I'm going to choose my database. The NR database, or the non-redundant database, is actually
a very very broad database
that does include redundant sequences
I'm going to use a little bit higher quality database A secondary database called the Reference proteins
and what that will do is
limit my output so I don't get duplicate sequences in my output
the third thing I want to show is the organism line I could limit my output to a specific
organism
or a a series of organisms. For instance, if I start to type out mammals you see that
mammals becomes one of my choices I could click on that
I'm going to limit to human sequences so I could just type human. I could use tha latin name as well, homo sapiens,
but human will work
Scrolling down just a little bit further
The blast button will start the
process
if I expanded this tab by clicking here
down below it will open some advanced parameters If you were more familiar with advanced blast
parameters you could adjust those. We're not going to do that in this exercise
I'm just going to go right ahead and click blast
and
I have got my output relatively quickly
you have some
basic parameters here at the very very top
along with a graphical view below that let me take you right down to the actual hits
hits are sorted by total score from highest the lowest but also by e or expect value
from lowest to highest
and
just in case
you're unfamiliar the six e minus twenty-one on this line
means six times 10 to the minus 21st
and what we're looking for is a very very low e value
anything below one times 10 to the minus 4
can be considered homologous or related to the sequence that we started with
so essentially
all of the sequences that I'm going to highlight
are relatives of that rat sequence
in the human genome
anything below that line we can't make any conclusion about because
the
aligned region is a little bit too short
so there are
about twelve homologues to that rat protein in human
and if I scroll further down I can actually see the alignment
and if you notice the first alignment
there are one hundred seventy nine amino acids that match exactly
so
there is a good chunk of this rat protein that exactly matches the human sequences
this is a nearly identical sequence
in fact of that one-hundred seventy-nine amino acid overlap
one-hundred seventy-nine of the amino acids are identical
this is exactly the same sequence
let me show you a sequence that's a little bit different just below it
and here we have
at the top on the top line is the query sequence
that's what we started with, that's our rat sequence
and here is that second best hit in human at the bottom
and then you see a series of letters and plus signs and spaces in between
any time that the sequence matches exactly
in the middle you'll see a capital letter so you'll see an R when there is an R against an R
and that happens hundred times so hundred out of a hundred seventy-nine amino acids
are identical or fifty five percent of the amino acids are identical
Now in addition there are some amino acids with plus signs For instance, there is a plus sign
between this V and the I
The V is valine and the I isoleucine
chemically they are very very similar so although it is not an exact match it is very very close chemically
so that's considered
similar
so if you notice under positives
it says one-hundred twenty-eight out of one-hundred seventy-nine and we know hundred are already
identical
so what that means is that there are twenty-eight more
that are similar enough
that we can call that a match so if I were explaining this I would say that these
two proteins
are fifty-five percent identical
and seventy-one percent similar
the last thing I want to show
is the Edit and Resubmit button
if you wanted to start over the back button isn't necessarily conduCive to saving your
previous search
Edit and Resubmit will
Take you back to the blast main page
exactly where you started
and you can make any adjustment
the you'd like to for instance
changing database
changing sequence
or maybe changing your output organism