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[MUSIC]

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This exercise is going to be on the
PsiBlast tool at NCBI.

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PsiBlast stands for position specific
iterated

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blast, which is a specific form of

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blast, designed to find distant homologs
of a particular protein or DNA sequence.

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Our objectives for this exercise include
setting up the parameters of PsiBlast,

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specifically to get the right threshold
for future iterations of PsiBlast.

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We will enter a protein sequence to run
the first

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iteration of PsiBlast, and then from those
results, we will

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be able to compile a group of sequences to
run

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a second iteration to find other members
of that protein family.

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Finally, we will be looking in more detail
how to

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interpret those results, which can be a
little bit tricky.

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First, we will retrieve the protein
sequence that we were going

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to use for PsiBlast, and that sequence is
available at NCBI.

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Specifically, we'll be using a transposase
from Pyrococcus furiosus.

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Pyrococcus furiosus is a hyperthermophile
that grows at 100 degrees Celsius.

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It comes from a volcanic vent.

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The organism was first isolated off the
coast of

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Italy from a volcanic vent on the
Mediterranean floor.

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Now I'm going to click FASTA to get the
actual sequence, and here is the sequence.

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What you might notice is that the first

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line is descriptive, describes the
transposes for Pyrococcus furiosus.

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And then below that are a series of
letters,

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which represent the amino acids in that
particular protein.

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Okay.

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You might notice that the top line, which
I have highlighted, is the description.

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The description says that this is a

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transposase from Pyrococcus furiosus, and
then below that

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are a series of letters, and these letters

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represent the amino acids in a particular
sequence.

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Now the the FASTA formatted sequence
includes both the amino acids

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sequence and the descriptions, so I'm
going to copy and paste that.

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And now I'm going to go and do the
PsiBlast search.

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To do that, I've got to go to the blast
page at NCBI,

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and you can Google that or type in NCBI,
for National

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Center for Biotechnology Information,
.NLM, for

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National Library of Medicine, .NLH, for
National

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Institutes of Health, .gov, and then slash
last.

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That will take me to the main blast page,
and I'm going to click protein blast.

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And if you notice, by protein blast,
you'll see a series of algorithms.

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The algorithms include, blast p, psi
blast, phi blast, and delta blast.

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So, we're going to click protein blast,
and the first thing I'm

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going to do is paste in the sequence into
the window.

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So there is my, the transposee is from
pyrococcus furiosus.

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Okay.

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Now I'm going to select PsiBlast.

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[BLANK_AUDIO]

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And I'm going to choose my database.

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My database, in this case, is going to be
Reference Proteins.

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It's a more high quality database than the
NR database.

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The NR database stands for non-redundant,

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but there are plenty of redundant
sequences.

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It's a huge database with a lot of low
quality sequences.

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We'll switch to the reference proteins,
and one thing I want to do

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before actually running the PsiBlast is
you'll

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notice algorithm parameters in the
lower-left corner.

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We're going to expand that window, and I'm
going to change the threshold.

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Scroll all the way down.

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You'll see the default PsiBlast threshold
is 0.005.

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I want to be a little more stringent than
that.

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I'm going to use 1 times 10 to the minus
4th to make sure

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that I include only members of the

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transposes family when I do the second
iteration.

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So I'm going to change that to 0.0001.

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Okay, so I have my protein sequence.

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I have my database.

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I selected PsiBlast, and I've changed my
threshold.

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One thing I think I want to do is limit to
a particular type of organism, so the goal

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of our exercise might be to find homologs
or

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relatives of this protein in a particular
type of bacteria.

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So I'm going to enter Lacto Bacillus,
which will give me lactic acid bacteria.

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Many different types, and you see that
that's

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taxonomic ID 1578, so I'm going to click
that.

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And now the results should come up
shortly.

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[BLANK_AUDIO]

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Okay, so here are the results, and you
notice

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that there are a series of alignments
better than

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threshold, and there are a whole bunch of
relatives

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of the pirachocas transposes in
Lactobacillus.

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And then you also see a series of proteins
that scored worse than the threshold.

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Now typically for blast, if a, if an e
value, is less than 1 times

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10 to the minus 4th, such as this one, 6
times 10 to the negative 5.

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We can consider that a homolog or a
relative of the protein that we entered.

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So everything above this line we can very,
very

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confidently say, yes, these are homologs
of that pyrococcus transposase.

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Anything below the line we're not quite as
sure of.

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[BLANK_AUDIO]

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Okay, but we see that there are some
transposases there.

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So what we can do is examine the results.

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Notice that almost all of these results
are from transposases.

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Even some of the ones that are worse than
threshold are transposases, but

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notice that everything above the line,
every protein above the line, has a check.

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On the right side in that check box, and
if you notice the header under

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worse for threshold says select for side

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list, anything below that threshold is not
checked.

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So what PsiBlast is about to do in the
second iteration is develop a

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position specific scoring matrix or possum
as we commonly call them.

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The possum is a mathematical
representation of the sequence that we

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started with and all of these relatives
that are above threshhold,

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and if I wanted to manually exclude a
protein, I say,

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oh, yeah, maybe that's not a relative, I
could do that.

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I could also take a sequence that's worse
than threshold.

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I'll actually take the second one here.

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It's a transposase.

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Its e value is 1 times 10 to the minus 4,
probably right on the border.

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So maybe I'll include that in the
PsiBlast, and now when I hit go, I'm going

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to run a second version of blast, a

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second iteration, except not with a query
sequence.

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Now with this possum, that mathematically
represents all the

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of the results in the first iteration of
blasts.

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Okay, and these results should come
shortly.

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Okay, now what we notice is, first of all,
a bunch of very, very strong e values.

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2 times 10 to the minus 122.

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I'll talk more about that in a second.

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You'll also notice that some protein
sequences are

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highlighted in yellow, and the sequences
that are

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in white, without the yellow highlighting,
are protein

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sequences that were found in the first
iteration.

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Any new sequence that was only found

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in the second iteration is highlighted in
yellow.

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And we see some new sequences with very,

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very strong e values, and I want to
caution you

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about that because the nature of the
second iteration's

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very different from the nature of the
first iteration.

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In the first iteration, we started with
the, transposase sequence for pyrococcus.

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And, when I ran that first iteration, I
said that anything with an e

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value of less than 1 times 10 to the minus
4 is a homolog.

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So, now I see e values much, much less
than 1 times

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10 to the minus 4, but let's remember, in
the second iteration.

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We're not running the query sequence from
pyrococcus.

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We're now running a possum that represents
both

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the pyrococcus sequence and a bunch of
other transposes.

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So these e values say that the possum was
matched very, very

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well, which suggests that it's probably a
member of that protein family.

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However, in some cases, a possum might not
quite accurately represent

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a protein family, maybe because a
nonrelative got into that mix,

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and it's usually best to treat these new
yellow sequences as punitive

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homologs, meaning that they should be
further checked for homology.

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And you notice, in this case, that almost
all of the sequences highlighted in yellow

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say transposase, which tells me that they

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are probably indeed members of that
protein family.

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So we can see that PsiBlast is a powerful
tool for finding potential homologs of

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protein or DNA sequences that might not be
found by the first iteration of blast.

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Blast is not 100% sensitive, so it will
not

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necessarily find all homologs in an
initial database search.

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Psi-blast is a powerful way to identify
potential homologs that may

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have been missed by the first round of a
blast search.

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This concludes our PsiBlast tutorial.

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For more information, please contact us

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in the Center for Biolotechnology
Information.

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Thank you.

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[MUSIC]