454 Sequencing

Summary

This 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

• 🌟 Next-generation sequencing technology, 454 sequencing, is fast and reliable, using fluorescence data to sequence DNA.
• 🧬 454 Sequencing is particularly effective for large genomes, such as the human genome, for whole genome sequencing.
• 🔬 The process begins with the fragmentation of the genome, typically using physical shearing to break down the DNA into smaller pieces.
• 🔗 Adapter ligation is a crucial step, involving two different adapters attached to the ends of the DNA fragments to facilitate sequencing.
• 🧵 The DNA strands need to be separated into single-stranded DNA to prepare for the sequencing process.
• 📿 Beads play a key role as a solid surface for attaching single-stranded DNA sequences with the help of adapter A.
• 🔄 Amplification of DNA is achieved without PCR, by using the complementary strand to produce more target DNA strands for sequencing.
• 🧬 The amplification process creates multiple copies of the DNA, which are then attached to the beads, facilitating the sequencing of multiple fragments.
• 🚀 The actual sequencing begins once the beads, loaded with DNA fragments, are placed into the sequencing machine's wells.
• 🔍 Fluorescently tagged nucleotides are added sequentially, and the fluorescence generated indicates the presence of specific nucleotides in the DNA sequence.
• 🖥️ 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

00:00

🧬 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.

05:03

🔬 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.

10:03

🌟 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.

15:05

🖥️ 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.

Keywords

💡DNA Sequencing

DNA Sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It is fundamental to modern biological research and medicine. In the video, DNA sequencing is the central theme, with a focus on next-generation sequencing technologies, particularly the 454 sequencing method, which is fast and reliable and relies on fluorescence data to sequence DNA.

💡454 Sequencing

454 Sequencing, also known as pyrosequencing, is a high-throughput method of DNA sequencing that is part of the next-generation sequencing technologies. The script explains that this technology is particularly effective for larger genomes like the human genome, and it involves several steps including fragmentation, adapter ligation, and amplification of DNA fragments.

💡Genome Fragmentation

Genome Fragmentation is the initial step in the 454 sequencing process where the entire genome is broken down into smaller, manageable pieces. The script mentions that this is typically done through physical shearing, which avoids chemical processes and results in a breakdown product of the genome that is then used for further sequencing steps.

💡Adapter Ligation

Adapter Ligation is a critical step in the 454 sequencing process where adapters are attached to the ends of the fragmented DNA. The script specifies that there are two different adapters, referred to as adapter A and adapter B, which are ligated to opposite ends of the DNA fragments, facilitating the subsequent steps of sequencing.

💡Single-Stranded DNA

Single-Stranded DNA refers to a DNA molecule that has been separated into individual strands, as opposed to the double-stranded DNA that is typical in a helical structure. The script emphasizes the importance of converting the double-stranded DNA fragments into single-stranded form before proceeding with the sequencing process.

💡Bead

In the context of the script, a Bead refers to an insoluble, large particle used in the 454 sequencing process. The single-stranded DNA with adapters is attached to these beads, which serve as a solid surface for the sequencing process. The beads are then loaded into wells for the actual sequencing to take place.

💡Amplification

Amplification in the script refers to the process of increasing the quantity of a particular DNA fragment to improve the accuracy of sequencing. The 454 sequencing method does not require PCR for amplification; instead, it uses a process where complementary DNA strands are produced and bind to other beads, creating multiple copies of the target DNA.

💡Fluorescence

Fluorescence is a property used in the 454 sequencing method to identify and measure the incorporation of nucleotides during sequencing. The script describes how each nucleotide is tagged with a different fluorescence color, and the intensity of the fluorescence signal corresponds to the number of nucleotides added at a particular position in the DNA sequence.

💡Primer

A Primer is a short nucleotide sequence that provides a starting point for DNA synthesis. In the script, primers bind to the adapter B region on the single-stranded DNA attached to the beads. The addition of nucleotides to the primer generates a fluorescence signal, which is used to determine the sequence of the DNA fragment.

💡Data Interpretation

Data Interpretation is the final step in the 454 sequencing process where the raw data from the sequencing machine is analyzed to determine the actual DNA sequence. The script mentions that software programs process the data from each fragment and attempt to overlap them to reconstruct the complete genome sequence.

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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