We will show you how to navigate genomic
database and use various bioinformatics
Tools to attract relevant
genomic information, including gene
sequence, its structure and function
annotation. This exercise will contribute
to better understand gene exploration in
the context of bioinformatics and its
potential use in animal genetics.
First, we will use the GenBank database. Until
in the search field, enter the name
RYR1 gene and designation
species, pig and give
look up. After a while, we
to display the results.
We see the information about the gene, here it is
but also about its
messenger RNA.
We see a unique gene number here
his ID.
And by clicking on the name of the gene, we get to
for more detailed information.
We can see that the gene is located on the
6. chromosome and we have information about two
genomic assemblies. One
older 105 annotations and one
newer 106. On
at the end of the table are the exact positions in
nucleotides within the genome of our
gene RYR1.
Below is a graphic representation of the position of
the RYR1 gene and genes,
that are adjacent to this gene,
right and left.
We can also see a graphic representation of the
gene structure. Including
positions of individual exons
and introns. Hover
Mouse cursor on this sequence
We see more details and the possibility
Download sub-sequences.
This is the sequence listed in the database
GenBank NCBI.
We also see another sequence of the same
gene listed in the Ensemble database.
We can read here about information such as
that it's a structural gene
What protein does it encode?
What is its length and more?
If you go below the links,
we click on the Fast record,
Fasta record,
We get into the sequence itself.
We can see that it really is
genomic sequence of whole-genome shotgun
sequence. From the genome
SusScrofa 11.1 in
positions of our selected region with Gene
RYR1.
We can see that the sequence is really long.
Ryr1 ge is one of the longest genes.
We will get more information on this sequence
by expanding the Fasta menu.
That offers us different possibilities,
download in various formats of this
sequence. When
We will go back to
information on the RYR1 gene
And we scroll a little lower we see
Express data information.
We can see that this gene is the most
expressed in skeletal muscle.
These are results from various
Publications.
Learn more about this
gene can be found below.
In particular, note that it is a gene
which involves the transport of calcium ions,
in the sarcoplasmic reticulum of the muscle
cells. They follow up on this
information about the protein about its functions.
Among other things, we read there that this applies to
pig stress syndrome.
And that it is basically about
protein with calcium function
Channel.
A little below are shown
Reference sequence information
transcripts, which in turn were described in
Literature. And stored in
GanBank database.
In the last part we can see
genomic or mRNA sequences,
which may be related or related to
gene sequence of the RYR1 gene in
Pig.
The last thing we can see is the link
to the protein RYR1 gene up to
Uniprot.
However, we will go back up and
click on the GenBank link
on the right.
Which brings us to the reference sequence
gene regions.
We find its unique number,
Furthermore, information on what kind it is.
And then what is the length of this sequence?
We see that over 118000 pairs
base. Well, and when
We move even lower, so we see
information about the gene.
How long is it and further about individual
sections that then form
transcribed mRNA, that is,
there are those coding sequences.
And when we click on mRNA.
We will see all these encoding,
exon sequences within the genomic
sequence.
And when we click on CDS
i.e. coding sequences.
In this way, we will only mark those
which parts of the sequence
sequences that will be
Translated into
Proteins.
And we see that really with the first triplet,
The first three bases
is ATG, which in mRNA language
means AUG, which is the initiation code
coding for the first amino acid in each
protein, methionine.
Because this gene is really very
long, also coding protein
of this gene is quite long.
We can also see its sequence here.
And that includes his identification number.
If we go back to the beginning
our searches,
We can click on the link to
sequence of messenger RNA.
Here, as you can see, it has over 15 thousand base pairs.
Again, this is a reference sequence,
which has its own unique number.
The reference sequence means that it has been
assembled from many mRNA subsequences
by various authors.
Here, too, we have various information, including
and about protein, about its
Sequence. And when
click on the CDS link, we get the coding
part of this sequence, that is, the one that
it encodes this protein. Because
it is mRNA, there are none
introns and we have 1 large unit.
Nevertheless, even here they are marked
individual exons from which this mRNA
is made up.
The second genomic database that we will
deal with it, is ENSEMBL.
It's again a public database,
in which there are vertebrates.
When we unfold the blind,
We can search for various
species, including humans, of course.
And we can find our
the species under study, the pig.
Enter the name in the search box again
or the abbreviation of our gene RYR1.
And in a moment we will see many results.
We will be interested in the reference
sequence.
Otherwise, we can find other sources here
information, sequence from
other authors, or even
individual pig breeds,
in which the same sequence was investigated.
On the next page in the results
we see similar information again, that is,
genome information where the gene is located -
Again, we see the 6th chromosome, in which
range with sequence,
Is our gene found?
And in the graphic display, we see again
results of various studies that
They cut the same gen.
But we will look at the offer
at the top left. Where
is a structure in which we choose a part of
Ontologies, and Looking
to information on
molecular functions.
Where do we actually see similar functions that
were in the Genbank database. Here it is
about the same features.
Next, we will look at biological
processes. Again, it is
same information. These are
protein that is involved in transport
Calcium
and transmembrane transport.
A processes at the cell level
it is again about
membrane protein, trans membrane
protein found in
sarcoplasmic reticulum.
In the Genetic variantion section, we will
to be interested in what has already been described
variability in this gene.
In tabular form, we can then
see a large number of
Mutations. In particular,
these are simple polymorphisms of the SNP,
but also deletions or advertisements.
And this from the beginning of the gene.
Furthermore, within introns and exons.
There is information about a specific position
of this polymorphism.
What is the alternation of nucleotides there,
And what type is it
variability.
The most interesting are the mutations that
are part of the exon, in particular
Misssence - variants that can
cause amino acid substitution.
We will be interested in the SNP that causes
amino acid swap at position 612.
This is a mutation that was described in
Fuji et al. published in
1991.
It is currently associated with
the sensitivity of pigs to stress;
so-called malignant hyperthermia.
In order to identify this mutation in
genome, we will use the sequence
described in this publication,
which we copy and paste into the new
file in a text editor.
Furthermore, from the genomic sequence,
format we download the sequence
Gene. And this
Copy the sequence to
Clipboard.
And because it will be really long, it will be
take a while to mark the whole
Sequence.
Again, we can insert this sequence into
separate text file to
They could continue to work with her.
As the main tool for finding an area
in the genome, where our
mutation, we will use the
BLAST, which performs local
aliment from a sequence from the publication
with genomic sequence.
Therefore, we check alignment 2 or more
Sequences. Until
upper window, we insert our genomic
sequence and to the bottom of this
sequence from
publication.
We leave the menu checked,
Highly simillar sekvences Megablast
and tick the offer to show us the result
appeared in a new window. Click
BLAST will start a search.
In the new window in the results, we can see in
table
alignments, in our case only one.
And if we click on it, we'll see
Assignment result
of our sequence from a publication with a whole gene
Sequences. Our sequence has 74
base pairs. And genome-wide
sequences start with
18176 based and
ends at 18249
Base. We copy
result in Word to make us better
worked with text.
We mark the place of the C/T mutation.
And this is the position of our SNP,
that causes amino acid substitution,
because it's 1 base in a triplet
TGC or CGC.
We know from the publication that the CGC variant
presents us with the original dominant
allele of a capital N, which causes
The position of the 615 amino acid is arginine.
Variant with T mutation or TGC
causes amino acid substitution into
Cysteine and that represents to us
recessive allele n, homozygous unit s
This mutation makes them susceptible to stress.
Thank you for your attention.