Hello! In this lecture, we will deal
with genomics in farm animals and, above
all, with sequencing. The lecture is
part of module 1, Animal Genetics.
The creation of this presentation was
supported by the ERASMUS+
KA2 grant within the
ISAGREED project. Innovation of the
content and structure of study programmes
in the field of aneimal genetics and food
resource management using
digitization.
DNA sequencing is a basic molecular
genetic method. The method makes it
possible to read genetic information,
which is stored in the form of sequence
of nucleotides in a DNA molecule.
In this figure, I present an overview
of the most important sequencing methods,
from the oldest to the most modern.
A chemical method based on the specific
cleavage of the DNA chain and
referred to by ta discoverers as
Maxam-Gilbert, was the first
sequencing method, but is not
longer used today. The
method discovered by Frederick Sanger,
after whom it is named, is still used
today. It is also referred to
as the dideoxy method
because it uses specially modified
nucleotides, so-called end
terminators. These are added
to the mixture with the normal
nucleotides, and if they are
incorporated into the chain, the
synthesis is terminated.
Pyrosequencing and sequencing by
hybridization are alternative
methods, the use of which is
currently small. Conversely,
next-generation sequencing
is very widespread and used due to its
high speed and sequencing capacity,
as it can also be used to sequence entire
genomes and is referred to
as second-generation
sequencing.
Methods based on the sequencing of single
DNA molecule referred to as
third-generation sequencing, are
increasingly being used. A
great advantage of sequencing is
that it enables simple and accurate
identification of a polymorphic
site.
Modern automatic sequencers are
also suitable for direct detection
of polymorphisms.
The principle of the method was
discovered in 1977
by the English biochemist Frederick
Sanger. The bases of Sanger sequencing
are dideoxynucleotides,
called ddNTPs or
end terminators.
ddNTP is a modified
nucleotide that has removed the OH
binding group on the 3rd carbon of
deoxyribose, which is necessary for
binding another nucleotide.
Thus, upon incorporation of the
ddNTP, chain synthesis
stops, producing a chain of the size
and color corresponding to the
corresponding nucleotide on the template.
The sequencing process consists of
several steps. It starts with sample
preparation, which is most often a
PCR product. This must be
purified to contain only template
DNA. Furthermore, we need to
know at least its approximate
concentration. The sequencing
reaction itself is the linear cyclic
enzymatic reaction that must
contain the listed components. The
sequencing primer defines the start of
sequencing, only one must be used,
not two as in PCR. The
reaction is catalyzed by DNA polymerase
inside the reaction buffer with the addition
of standard dNTPs
and additionally ddNTPs.
This is followed by purification of the
sequencing reaction when free labeled
ddNTPs must be removed.
The mixture of fragment is divided by
capillary electrophoresis into sequencers
and the result evaluated.
Currently, a variant known as 4-colour
terminator sequencing is routinely
used. Individual termination
nucleotides are marked with fluorescent
colours according to the heterogeneous
base they carry, or which nucleotide
on the template they pair with.
By the action of the polymerase, chains
complementary to the template are
synthesized, while usually normal
dNTPs are incorporated
and the chain is lengthened, but
occasionally ddNTPs
are also incorporated, which
then terminates the synthesis of the
chain. The resulting fragment
is labelled with a fluorescent colour
that corresponds to the nucleotide at the
appropriate position of the template.
A mixture of single-chain molecules of
different lengths and different colours is
formed. In order to determine at
which position which base occurs, an
accurate electrophoretic separation of
this mixture of fragments must take
place. To do this, it
uses fluorescent capillary
electrophoresis, which is the basis of
genetic analyzers or sequencers.
This device can not only divide and sort
the fragments according to size, but
also directly read the sequence of
nucleotides based on different coloured
peaks. E.g.
the 100 nucleotides
long chain emits green light,
i.e. there was adenine in position
100 of the template, the
chain 101 long
lights up red, i.e. in
position 101 was
thymine etc.
The Sanger method is the most widely used
sequencing method. Among its
advantage are the reading of relatively
long DNA chains
(approximately 800 bp)
and at the same time high accuracy and
reliability. If we need to know the
sequence or detect a mutation in a
specific section in genome in one or two
individuals, it is the most
cost-effective option. Compared to
NGS methods, however, the
performance and price per one
sequenced base is very high. So the
method is not suitable for sequencing
large section of the genome or even
entire genomes.
For whole genome sequencing, it is
more advisable to use another method,
the so-called next generation sequencing,
NGS for short. The
method is also referred to as second
generation sequencing. The method
is based on DNA fragmentation
and sequencing of short fragments, but
in huge quantity at the same time, and
is thus referred as a massively
parallel sequencing. The method
enables a generally very high sequencing
capacity. However, you can choose
sequencer variants with a capacity
according to your needs from 1
gigabase to 8 terabases.
Common NGS sequencers include Illumina
devices. The basis of the method is the
so-called bridge PCR and
sequencing during synthesis using
four-colour fluorescence. The
actual sequencing takes place in
clusters of the same sequence - the
corresponding spot lights up in colour
according to the base. Section
from 150 to 300
nucleotides in size are sequenced.
A more detailed explanation is beyond the
timeframe of this lecture, and I
recommend watching videos on the internet
for those interested in understanding
this rather complex method.
Next-generation sequencing is today a
widely used tool in genetics for
both research and diagnosis. The
high capacity of the methods makes it
possible to obtain sequences of
even large genomes of animals,
e.g. mammals, the size of which is around
3 billion nucleotides,
within a few hours to days, which
would take several years of work using
the Sanger sequencing method. It is
possible to obtain the entire genetic
information of an individual in this
way. It therefore allows
finding a large number of new or
detecting all no polymorphisms and
mutations. However, there is a
problem with the processing and
evaluation of large amounts of data,
which is why it is necessary to use very
powerful bioinformatics
tools. Whole-genome data are
stored in the genome databases of
individual organisms, and can be
compared with a specific sample.
Then methods is also important for
sequencing an individual,
e.g. patients, which is the
field of personal genomics.
Sequencing techniques based on the
sequencing of a single molecule are
referred to as 3rd generation
sequencing. The advantage is long
reads, in the case of de novo sequencing,
up to seven kb.
This makes it possible to correctly
identify variants on one chain,
the so-called haplotypes, which reading
of short sequencing is the second
generation, does not allow.
The method is gradually being improved,
as the reading error rate is still higher
compared to the two previous generations.
A method known as single molecule
real-time sequencing from Pacific
Bioscience or Nanopore sequencers from
Oxford Nanopore is available.
The results of whole-genome sequencing
are stored in genomic databases and
often freely available. For
example, the NCBI Internet
database allows viewing
down to the sequence level of
individual gene nucleotides.
Another practical use of sequencing in
animals is species identification.
Molecular taxonomy with so-called DNA
barcoding, which makes it possible to
identify a species where it is not
possible with the classical method,
e.g., in insect larvae.
A fragment of the gene for cytochrome
C oxidase 1, which is located
in mitochondrial DNA, is used for
this purpose. This
fragment is usually sequenced by
Sanger method, and species
identification is performed by
comparison with determined
sequences stored in the Bold or Blast
databases. The
advantage of mitochondrial
DNA is more copies, i.e.
greater amount of DNA per amount
of biological material, and higher
stability. It is also used for the
determination of older or museum
samples. The
disadvantage is the possibility of
contamination with a bacterial genome
(Wolbachia, etc.).
This image shows a sample of a
specific sequence obtained by
mitochondrial DNA sequencing
of an old museum butterfly specimen.
By inserting the sequence into the
Internet alignment search tool
called Blast, it was
possible to identify the species
based on homology.
The lecture is finished. I believe
that you have understood the basic
principles of one of the most important
molecular-genetic methods - sequencing.
I also recommend watching the follow-up
presentation: Laboratory examples.
Thank you for your attention.