454 Sequencing
Summary
TLDRThis video tutorial from Shaman's Biology delves into the advanced technology of 45 for DNA sequencing, a fast and reliable method belonging to next-generation sequencing. It outlines the process involving genome fragmentation, adapter ligation, and attachment to beads, followed by amplification and loading into wells. The video explains how fluorescence data is used to sequence DNA, with a focus on larger genomes like the human genome. It concludes with the interpretation of data to achieve the complete genome sequence, offering viewers insight into modern DNA sequencing techniques.
Takeaways
- π Next-generation sequencing technology, 454 sequencing, is fast and reliable, using fluorescence data to sequence DNA.
- 𧬠454 Sequencing is particularly effective for large genomes, such as the human genome, for whole genome sequencing.
- π¬ The process begins with the fragmentation of the genome, typically using physical shearing to break down the DNA into smaller pieces.
- π Adapter ligation is a crucial step, involving two different adapters attached to the ends of the DNA fragments to facilitate sequencing.
- 𧡠The DNA strands need to be separated into single-stranded DNA to prepare for the sequencing process.
- πΏ Beads play a key role as a solid surface for attaching single-stranded DNA sequences with the help of adapter A.
- π Amplification of DNA is achieved without PCR, by using the complementary strand to produce more target DNA strands for sequencing.
- 𧬠The amplification process creates multiple copies of the DNA, which are then attached to the beads, facilitating the sequencing of multiple fragments.
- π The actual sequencing begins once the beads, loaded with DNA fragments, are placed into the sequencing machine's wells.
- π Fluorescently tagged nucleotides are added sequentially, and the fluorescence generated indicates the presence of specific nucleotides in the DNA sequence.
- π₯οΈ Data interpretation involves processing the fluorescence measurements to determine the sequence of the DNA fragments and ultimately assemble the whole genome sequence.
Q & A
What is 454 sequencing and why is it considered a next-generation sequencing technology?
-454 sequencing, also known as pyrosequencing, is a high-throughput DNA sequencing method that is part of the next-generation sequencing (NGS) technologies. It is known for its speed and reliability, and it uses fluorescence data to sequence DNA. It is particularly effective for whole genome sequencing of larger genomes, such as the human genome.
What is the first stage in the 454 sequencing process?
-The first stage in 454 sequencing is the fragmentation of the genome. This is typically done using physical shearing to break down the genome into smaller pieces without using any chemical processes.
Why is adapter ligation an important step in 454 sequencing?
-Adapter ligation is crucial in 454 sequencing because it involves attaching two different adapters to the ends of the fragmented DNA. These adapters are single-stranded DNA sequences that facilitate the subsequent steps of sequencing by providing a binding site for the sequencing process.
How does the process of making DNA single-stranded relate to the 454 sequencing method?
-After genome fragmentation, the DNA obtained is double-stranded. In 454 sequencing, it is necessary to separate the strands to make them single-stranded. This is important because the adapters are ligated to the single-stranded DNA fragments, which are then used in the sequencing process.
What role do beads play in the 454 sequencing process?
-In 454 sequencing, beads are used as a solid surface to which the single-stranded DNA fragments, with adapters ligated at both ends, attach. The beads are constructed to allow a section of nucleotide sequence to bind to them, facilitating the attachment of the DNA fragments.
How is DNA amplified in the 454 sequencing process without using PCR?
-DNA amplification in 454 sequencing is achieved through a process where the nucleotide sequences are added to produce complementary structures. This is done repeatedly to generate multiple copies of the target DNA strands, which are then attached to the beads, without the need for a PCR process.
What is the purpose of loading the beads into wells in the 454 sequencing process?
-Loading the beads into wells is a preparatory step before the actual sequencing begins. The wells are small volumetric areas where the beads, now filled with DNA fragments, are placed. This allows for the organized and controlled sequencing of multiple DNA samples simultaneously.
How does the addition of primers and nucleotide sequences contribute to the sequencing process in 454 sequencing?
-Primers are added to bind with the adapter B region on the DNA fragments attached to the beads. The addition of nucleotide sequences generates a specific fluorescence each time a nucleotide is incorporated. This fluorescence is used to determine the sequence of the DNA, as each nucleotide corresponds to a different color.
What is the significance of fluorescence in determining the DNA sequence in 454 sequencing?
-Fluorescence is key in 454 sequencing as it allows for the detection of incorporated nucleotides. Each type of nucleotide is tagged with a different fluorescence color. The intensity and color of the fluorescence generated indicate the presence and number of specific nucleotides in the sequence, which helps in determining the DNA sequence.
How does the 454 sequencing process interpret the fluorescence data to obtain the DNA sequence?
-The fluorescence data obtained during the sequencing process is analyzed by software programs. These programs process the data for each fragment, overlap the sequences, and assemble the complete sequence of the whole genome based on the fluorescence intensity and color patterns.
Outlines
𧬠Introduction to 45 DNA Sequencing
This paragraph introduces the topic of 45 DNA sequencing, a next-generation sequencing technology. It's highlighted as a fast and reliable method that uses fluorescence data to sequence DNA, particularly for larger genomes such as the human genome. The process starts with the fragmentation of the genome through physical shearing, followed by the ligation of two different adapters to the DNA fragments, which is essential for driving the sequencing process. The paragraph also describes the preparation of single-stranded DNA with adapters ligated at both ends, which is then attached to beads, setting the stage for the sequencing process.
π¬ DNA Sequencing Process and Bead Attachment
This section delves deeper into the 45 DNA sequencing process, focusing on the attachment of DNA to beads. It explains the role of adapter A in binding the DNA to the bead's solid surface and the subsequent amplification of DNA to increase sequencing accuracy. The process does not require PCR for amplification; instead, it uses the complementary strand to produce more target DNA. The paragraph also describes how multiple DNA fragments are handled, each undergoing the same steps of single-stranded DNA preparation, attachment to beads, and amplification, ultimately leading to the loading of these bead-filled fragments into wells for sequencing.
π Sequencing Reaction and Data Interpretation
This paragraph outlines the actual sequencing process, starting with the addition of primers that bind to adapter B on the target DNA. The sequencing reaction involves the addition of nucleotides, each tagged with a specific fluorescence color, which generates a fluorescence signal upon binding. The intensity of this fluorescence indicates the number of nucleotide repeats in the sequence. The paragraph details how the fluorescence data is used to determine the presence of specific nucleotides and deduce the complementary sequence, which in turn reveals the actual DNA sequence of the target fragment. The process is repeated for different nucleotides to sequence the entire fragment.
π₯οΈ Data Processing and Complete Genome Sequencing
The final paragraph discusses the post-sequencing steps where software programs process the data obtained from each fragment. These programs attempt to overlap the sequences to assemble the complete genome sequence. The paragraph emphasizes the comprehensive nature of the 45 DNA sequencing process, from the initial fragmentation and adapter ligation to the final data interpretation, and encourages viewers to like, share, and subscribe for more educational content on the topic.
Mindmap
Keywords
π‘DNA Sequencing
π‘454 Sequencing
π‘Genome Fragmentation
π‘Adapter Ligation
π‘Single-Stranded DNA
π‘Bead
π‘Amplification
π‘Fluorescence
π‘Primer
π‘Data Interpretation
Highlights
Introduction to 454 sequencing, a next-generation DNA sequencing technology that is fast and reliable.
454 sequencing relies on fluorescence data for DNA sequencing.
The technology is particularly effective for whole genome sequencing of larger genomes like the human genome.
The first stage of 454 sequencing involves the fragmentation of the genome using physical shearing.
Adapter ligation is the second stage, requiring two different adapters to be attached to the DNA fragments.
The importance of adapter ligation for driving the DNA sequencing process.
The necessity of converting double-stranded DNA to single-stranded DNA for the sequencing process.
The role of adapters in attaching single-stranded DNA to solid surfaces like beads.
Beads are insoluble particles used to attach and amplify DNA sequences.
The amplification process in 454 sequencing does not require PCR.
Amplification produces multiple copies of the target DNA for more accurate sequencing results.
Loading the beads filled with DNA fragments into wells for the actual sequencing process.
The sequencing process begins with the addition of primers that bind to adapter B.
The use of fluorescence to identify the presence of specific nucleotides in the DNA sequence.
The interpretation of fluorescence data to determine the complementary and actual DNA sequence.
The process of sequencing multiple DNA fragments simultaneously from different wells.
The final stage of 454 sequencing involves data interpretation to assemble the complete genome sequence.
The video aims to help viewers understand the process of next-generation sequencing, specifically 454 sequencing.
Transcripts
welcome back friends welcome to another
video from shamans biology and in this
video tutorial we'll be talking about
the four five for DNA sequencing we have
been talking about DNA sequencing for a
long time and this is one of the very
new kind of DNA sequencing technologies
belonging to the next generation DNA
sequencing technology is known as four
five bore DNA sequencing now four or
five for TNA sequencing is fast and it's
also reliable and it depends on
fluorescence data to sequence the DNA
now what is this for five for DNA
sequencing in this DNA sequencing it's
only used for larger genomes for example
human genome or any other organisms
whole genome sequencing is very
effective the idea is you have the whole
genome which is much more complex bigger
and the first stage of this sequencing
is the fragmentation of the genome see
fragment the genome using physical
sharing most of the time because you
don't use any chemical process their
physical sharing will give us the
breakdown product of that genome so once
you have the breakdown product of the
genome then the second stage is the
ligation of adapters because in this for
five for DNA sequencing we have two
different adapters to be ligated to the
fragments of the genome and this is
called the LA this is known as the
adapter ligation and this is required so
two different adapters say adapter one
adapter a and let's say this one adapter
be two different adapters are ligated in
two opposite terminal of the DNA so let
us say here this is the adapter be this
up
and adapter a also now this adapter
ligation is very important because this
adapter will help us to drive the whole
process of DNA sequencing because you
know the adapters that we add they are
nothing but DNA sequence single-stranded
DNA sequence now we begin with the
genome fragmentation once we fragment
the genome the DNA sequence that we get
is also double-stranded so after that we
need to separate the strands of the DNA
to make it single-stranded that is very
very important to make it as a
single-stranded DNA okay so we have the
single-stranded DNA
once we have the single-stranded DNA we
add the adapter or sometimes we can also
add the double stranded adapter at the
very beginning where the DNA is also
double standard in that case let us say
after the fragmentation of the DNA we
get this it said this is the DNA and
after that we add the adapter as double
stranded in both the ends let's say this
is the adapter a and this is the adapter
be neither the way we can do that okay
we can add it as a double standard or we
can take the double stranded DNA make it
single-stranded we separate the strands
then we can also add that up to us so if
we go like this say the double stranded
fragment DNA and we add the adapter
after the ligation of the adapter we
will separate the DNA strands so
ultimately we will need the separated
DNA strands for the whole process so now
we have the single stranded DNA here
where we have two adapters ligated at
the terminal here and here okay so this
is the preparation that we require at
the end so this single stranded DNA with
two adapters ligated at both the ends
now it is taken and we use this to be
attached to the Beeb's okay because we
have beads that are present now what
does this beads
these beads are insoluble molecules
these are large particles okay made up
with some molecules which are not
interacting with any other chemicals and
enzymes that we use here so you have
this beads small particle like beads
now the beads are constructed in such a
way so that we can add a small section
of nucleotide sequence single-stranded
nucleotide sequence at the app
surrounding the beads okay we input all
those single standard sequence and the
mean so let me draw the exact structure
of the bead say here so bead will look
something like this
okay the particle and single standard
nucleotide sequences are added covered
covering the beads so we have this now
the role of adapter a remember this role
of adapter a this one red one is to
attach with the sequence that is present
in the bead that is why we add this
adapter because we want this DNA
sequence to be fixed properly to a solid
surface
now bead is a solid surface right and
beads also carry this single standard
DNA sequence which is complementary to
the adapter a sequence so now adapter a
sequence can easily bind okay with this
bead and then rest of the DNA is placed
okay
for example we only draw one this is the
condition and obviously adapter B is
present here this is a scenario so once
the bead is attached with the DNA
sequence DNA fragment then the this is
probably the fourth process the fourth
step is to amplify this DNA because you
know we want to run as many times as we
can for the gene sequencing because if
you run more times the gene sequencing
results will be more accurate right so
we want every single fraction of this
fragmented DNA to be multiplied
be amplified and to be run multiple
times to get better sequencing result
more accurate sequencing results so we
once we attach that after that and start
adding nucleotide sequences start adding
the nucleotide sequences and the
nucleotide sequence will produce the
complimentary structure for this deal
okay so every time it will produce the
complementary structure once it produces
the complementary structure then again
it will go and bind to someplace else
next and then again that their
complimentary structure will go so the
idea is always this this same way so it
will produce the complementary structure
of it then we take this DNA
complimentary let's say this whole DNA
and it will go and bind to some other
beads with the complimentary nature and
then again it will produce this
complimentary DNA so it will go on and
on and on to produce the multiple copies
of this DNA to be amplified so here
actually we do not require PCR process
for the amplification of the target DNA
okay because you know after it
replicates it will produce a
complimentary stand now if you use this
complementary strand it can produce our
target DNA gain so use this
complementary strand to produce more and
more target DNA's in the whales okay
because once we produce these beats this
whole thing is going on in a tube okay
in a tube or a big well big giant well
so once you produce this complimentary
DNA we run that amplification process we
just add all the nucleotide sequences
time to time and also the DNA polymerase
which will easily produce the
complimentary DNA strand the idea here
is to produce more complimentary DNA
Stanmore strand of our interest because
we want to run those strand in the
sequencer more often okay so by this way
we produce multiple numbers of our
target DNA strands so once all those
strands are produced remember once all
those strands are produced then it's
time to attach those strands to the
beads because you know all of them
contains this result omit this part of
the a
dr. a so with the help of adapter a all
those structures all those structures
will start at it all those DNA will
start adding so ultimately what will
happen we have the DNA attached the
target DNA which is to be sequence
attached ok and at the end we have that
adapter B this is the rep train this is
how the whole beads are filled now this
is the time where we add so this thing
is going on for not only one fragment
but for each of the fragments that are
present okay because there will be
multiple DNA fragments so for each of
the fragments we do the same thing okay
and once they produce this
single-stranded DNA fragments and
they're attached to the beads now the
beads are filled with such DNA fragments
together then what we do we take the
beads and we load them into wells okay
there are small plates it's they're not
that big very smaller tiny plates tiny
wells Wells means small volumetric area
where we put all the beads covering the
target DNA there because once you put
into the wells the well is inserted into
the actual sequencing machine till now
whatever thing is going on is a pre
sequencing process once everything is
ready then we load them into the gel not
gel wells upon loading into the wells
when we put the wells into the sequencer
then the actual sequencing will begin
knowing every sequencing technology you
know every next-generation sequencing
technology this is very common step the
first step is the fragmentation of the
genome second stage is the adapter
ligation and the third stage is the
attachment of the target DNA to the
beads and fourth stage is loading the
bits into the well and the fifth and
final stage is the running of the
sequence and the sixth stage which you
can final not the fifth the fifth stage
is the interpretation of the data to get
the sequence so these are the major six
different phases of this whole
sequencing process most of the new next
generation sequencing process for five
four is one of them so once you load
them into the gel so this is the
condition now we want to check the
actual sequence the sequence of this
black fragment this is the target
fragment
so for that what we do we add primers we
add primers the primers will bind with
this adapter B region okay that is why
the adapter B is required so the primer
is attached to the adapter B and then
what we do we extrapolate this primer
okay because you know upon adding the
primer we have a free three prime
hydroxyl group at the end where the new
nucleotide sequences if added one after
another each time a nucleotide sequences
added a specific fluorescence is
generated okay so we can tag each of
those nucleotide sequences with
different fluorescence color green blue
red yellow different colors so each time
a fluid time a nucleotide sequence is
added it will generate a fluorescence or
we can allow one fluorescence color and
what we can do we can run the whole
process for each nucleotide sequences at
a time let us say we add the first try
it for an equal ater sequence thymine so
what we know timing is stacked with the
fluorescence so whenever thymine is
present in the complementary strand
wherever the atom is present in the
complementary strand thymine we pair
with it
and upon an addition of the thymine with
adenine it will generate the
fluorescence okay or something okay
because after each time let's say we add
the timing and let us say here
consecutive three adenine sir present
and let us say here no atoms are present
so what will happen priming is also here
so what will happen
timing will not bind with guanine here
so no attachment but yet three thymines
will pair there will be three different
attachments now after this whole binding
state will wash it completely after the
wash what we get we get only the data
from the bound timing as a fluorescence
measurement okay so we can get the
fluorescence as three timings are
present the degree of fluorescence will
be 3x three times more if one time is
present it will be only of one type if
there is no timing there won't be any
fluorescence measured from that point so
by this way we can check wherever there
is a space
nucleotide sequence is present or not in
the target DNA okay and if we know the
complementary sequence we are obviously
know the actual sequence that that is
the complementary of it so this is the
idea of sequencing now after the time
let us say we go for guanine sequence so
wherever if there is a c1 in you'll
attach a gate of fluorescence if there
is no c1 you will not attach no
fluorescence more than one guanine
cytosine to guanine will consecutively
added let's say in to cytosine is
present to go on insulated so the data
the degree of fluorescence that we get
will be twice to x2 times more so by
this way we can get a graph what kind of
gruffly expecting here we expect the
graph from the beginning a balance a
basic level of fluorescence is always
there but now after that let us say this
is for the guanine okay and this is let
us say for - it's for one say like this
okay this is for three let us say this
is the base level for example say this
is cytosine adenine that means guanine
is one so the intensity of the
fluorescence is once
so one guanine is present at the point
after that we have a three C so three
cytosine residues because three means
the intensity stripped three times more
so obviously three is present in this
case it is only two so by this way we
can understand the complementary
sequence now if you know the
complementary is guanine obviously the
actual target sequence will be C
cytosine if this is the complementary
cytosine the actual target sequence will
be go on if the complementary is added
in the actual sequence will be hanging
so now this is the sequence that we want
to find out by this way we can find out
the sequence of the complete fragment
eyes DNA at a time from different wells
so once we get the idea of the fragment
eyes DNA sequence for different wells
then if the data will be fade to a
monitor CPU through which the CPU
process there are software programs
designed for this will process the data
for each of the fragments and they will
try to overlap those fragments to get
the actual complete sequence of the
whole genome and this is how the whole
process works for a45 for sequencing I
hope this video helps you to understand
the next generation sequencing like four
five four sequencing if you like this
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