Ion torrent sequencing

Summary

This video tutorial delves into Ion Torrent Sequencing, a rapid and reliable DNA sequencing technology by Life Technologies. It explains the process of fragmentizing DNA, attaching it to beads, and sequencing by detecting hydrogen ion release upon nucleotide addition. The tutorial highlights the technology's speed, scalability, and high accuracy compared to Illumina sequencing, with the ability to sequence large genomes in just a few hours. The method's simplicity and cost-effectiveness make it an exciting advancement in the field of genomics.

Takeaways

• 🌟 Ion Torrent Sequencing, also known as Ion Proton Sequencing, is a fast and reliable DNA sequencing technology from Life Technologies.
• 🔬 The technology is based on the detection of hydrogen ion (proton) release during the polymerization stage of DNA sequencing.
• 🧬 The process involves fragmentizing the genome sequence, attaching adapters, amplifying the fragments, and attaching them to beads.
• 📐 DNA sequences are attached to single-stranded DNA on the beads, which are then loaded into tiny wells on a microchip.
• 💡 The sequencing chip functions like a semiconductor chip with millions of small wells, each capable of holding a bead with a DNA fragment.
• 🚀 The sequencing process is incredibly fast, taking only 2 to 3 hours for large DNA sequences, compared to 30 to 48 hours for other Next Generation Sequencing technologies.
• 🔍 The Ion Sensitive layer detects the release of hydrogen ions, which causes a pH change in the solution, allowing for the identification of each base added to the growing DNA chain.
• 📈 The technology scales up easily, with chips starting from 10 MB and now having a 100 GB capability, allowing for the sequencing of billions of DNA fragments simultaneously.
• 🛠️ The process is straightforward and does not require fluorescence, making it a clean and efficient method for DNA sequencing.
• 📊 The data obtained from the sequencing is highly reliable and can be aligned using complex algorithms to reconstruct the full genome sequence.
• 💰 While the technology may be expensive, the time and cost savings from the rapid sequencing process can offset the initial investment.

Q & A

What is Ion Torrent Sequencing?

Ion Torrent Sequencing, also known as Ion Proton Sequencing, is a Next Generation DNA sequencing technology developed by Life Technologies. It is known for its speed and reliability in generating DNA sequence data.

How does the speed of Ion Torrent Sequencing compare to other sequencing technologies?

Ion Torrent Sequencing is significantly faster than other technologies like Illumina sequencing. While Illumina might take 30 to 48 hours, Ion Torrent can complete the process in just 2 to 3 hours.

What is the basic principle behind Ion Torrent Sequencing?

The technology relies on the detection of hydrogen ions released during the polymerization stage of DNA sequencing. Each addition of a nucleotide to the growing chain releases a proton, which is detected to infer the incorporation of the corresponding base.

What is the role of adapters in Ion Torrent Sequencing?

Adapters are added to the ends of fragmented DNA sequences to facilitate their attachment to beads. The adapters are complementary to the single-stranded DNA sequences present on the beads, allowing the DNA fragments to bind effectively.

How are DNA fragments attached to beads in Ion Torrent Sequencing?

After adding adapters to the fragmented DNA, the DNA sequences are made single-stranded and then bind to the beads, which have been prepared with a complementary DNA sequence to the adapters.

What is the significance of the microchip in Ion Torrent Sequencing?

The microchip contains millions to billions of tiny wells, each capable of holding a bead with attached DNA fragments. This setup allows for parallel processing of a vast number of DNA sequences simultaneously.

How does the Ion Sensitive layer contribute to the sequencing process?

The Ion Sensitive layer detects the presence of positively charged ions, specifically protons, released during nucleotide incorporation. Changes in pH due to proton release are monitored, allowing for the identification of incorporated bases.

What is a 'base call' in the context of Ion Torrent Sequencing?

A 'base call' refers to the detection and recording of a specific base incorporation during sequencing. Each pH change, corresponding to a proton release, is counted as a base call, indicating the presence of a particular base.

How does Ion Torrent Sequencing handle the four different DNA bases?

The sequencing process involves adding each type of nucleotide one at a time. For example, adenine (A) is added first, and wherever it pairs with thymine (T) on the template strand, a proton is released, which is detected as a base call for A.

What are the advantages of Ion Torrent Sequencing over other methods?

The major advantages include its speed, reliability, and the ability to handle large-scale sequencing with minimal time and complexity. It also does not require fluorescence, making the process cleaner and potentially more cost-effective.

How does the scaling of Ion Torrent Sequencing chips impact its capabilities?

The scaling up of the chips, from an initial 10 MB to a current 100 GB capability, allows for the processing of billions of DNA fragments at once, significantly reducing the time required for large genome sequencing projects.

Outlines

00:00

🌟 Introduction to Ion Torrent Sequencing

This paragraph introduces the topic of Ion Torrent Sequencing, also known as Ion Proton Sequencing, a high-speed and reliable DNA sequencing technology developed by Life Technologies. It emphasizes the technology's key advantage of speed and reliability compared to Illumina sequencing. The process involves fragmentizing the genome, attaching adapters, amplifying DNA fragments, and attaching them to beads. The sequencing relies on the detection of hydrogen ions released during the polymerization stage of nucleotide sequencing, which is a unique chemical aspect of this technology.

05:01

🧬 DNA Fragmentation and Bead Attachment in Ion Torrent Sequencing

This section delves into the specifics of DNA fragmentation and the subsequent steps in the Ion Torrent Sequencing process. DNA is broken down into smaller parts, adapters are added to both ends, and the fragments are attached to insoluble beads. The beads, each containing a specific single-stranded DNA sequence, are prepared with a complementary sequence to the adapters. This allows the fragmented DNA to bind to the beads, setting the stage for the sequencing process, which takes place in tiny wells on a microchip, similar to the pixels in a digital camera sensor.

10:02

🛠 The Sequencing Chip and pH Detection in Ion Torrent Sequencing

The paragraph explains the role of the sequencing chip in Ion Torrent Sequencing. Beads with attached DNA sequences are loaded into wells on the chip, which is then covered by an Ion Sensitive layer capable of detecting the presence of positively charged ions, specifically hydrogen ions. During the sequencing process, the addition of each nucleotide releases a hydrogen ion, which causes a change in pH. This pH change is monitored and recorded as a base call, indicating the presence of a specific base. The chip functions like millions of tiny pH meters, recording base calls as the sequencing unfolds.

15:04

🚀 Advantages and Process of Ion Torrent Sequencing

This final paragraph highlights the advantages of Ion Torrent Sequencing, such as its ability to handle large genomes with high throughput and speed. The chip's capacity has increased from 10 MB to 100 GB, allowing for the simultaneous sequencing of billions of DNA fragments. The process is faster than other Next Generation Sequencing technologies, taking only 2 to 3 hours compared to 30 to 48 hours for Illumina sequencing. The sequencing process involves running the machine for each nucleotide one at a time, using pH changes as a signal for base calls. The technology is cost-effective in terms of time saved and does not require fluorescence, making it a clean and efficient method for DNA sequencing.

Keywords

💡Ion Torrent Sequencing

Ion Torrent Sequencing, also known as Ion Proton Sequencing, is a type of Next Generation Sequencing (NGS) technology developed by Life Technologies. It is characterized by its speed and reliability. The process involves the detection of hydrogen ions released during the polymerization of nucleotides, which is a key aspect of the sequencing method described in the video. It is integral to the video's theme as it represents the main topic being discussed.

💡Hydrogen Ion

A hydrogen ion, or proton, plays a central role in Ion Torrent Sequencing as the release of these ions during nucleotide addition is detected to determine the sequence of DNA. In the video, it is explained that the release of a hydrogen ion corresponds to the addition of a nucleotide, which is essential for the sequencing process.

💡Phosphodiester Bond

The phosphodiester bond is a chemical link formed between nucleotides in a DNA strand. In the script, it is mentioned that during the sequencing process, a new nucleotide's hydroxy group attacks the alpha phosphate, leading to the release of two phosphate groups as pyrophosphate and the formation of a phosphodiester bond. This concept is crucial for understanding the chemical basis of DNA sequencing.

💡Adapter

Adapters are short DNA sequences added to the ends of DNA fragments to facilitate their attachment to beads in the sequencing process. The video explains that adapters are necessary for binding the DNA to the beads, which is a common step in NGS technologies, including Ion Torrent Sequencing.

💡Bead

In the context of the video, beads refer to insoluble molecules to which DNA sequences are attached. Each bead contains a specific type of single-stranded DNA sequence, and these beads are loaded into tiny wells on a microchip for the sequencing process. Beads are a fundamental component of the Ion Torrent Sequencing method.

💡Microchip

A microchip, as described in the video, is a small semiconductor chip divided into millions of tiny wells, each capable of holding a bead with a DNA sequence. The microchip is a critical part of the Ion Torrent Sequencing platform, where the actual sequencing reactions take place.

💡Ion Sensitive Layer

The Ion Sensitive Layer is a component of the Ion Torrent Sequencing system that detects the presence of positively charged ions, specifically hydrogen ions. It is akin to a sensor that monitors changes in pH due to the release of hydrogen ions during nucleotide addition, which is a direct measure of DNA sequence determination.

💡Base Call

A base call in sequencing refers to the identification of a specific nucleotide at a certain position in the DNA sequence. In the video, it is explained that the Ion Sensitive Layer counts the release of hydrogen ions as base calls, indicating the presence of a particular nucleotide, which is a fundamental aspect of data collection in Ion Torrent Sequencing.

💡Sequencing Chip

The sequencing chip is the heart of the Ion Torrent Sequencing system. It is loaded with beads containing DNA sequences in the wells. The video emphasizes the chip's large capacity, which allows for the sequencing of billions of DNA fragments simultaneously, contributing to the speed and efficiency of the technology.

💡Deoxyribonucleotide Triphosphate (dNTP)

dNTPs are the building blocks used in DNA synthesis, with each type corresponding to a specific nucleotide base. In the video, the addition of a specific dNTP (e.g., dATP for adenine) is described as a step in the sequencing process, where it binds to the complementary base on the growing DNA strand, resulting in the release of a hydrogen ion.

💡PCR Amplification

PCR (Polymerase Chain Reaction) amplification is a technique used to make multiple copies of a DNA segment. The video mentions the use of PCR to increase the quantity of DNA fragments, ensuring that there are enough copies for accurate sequencing. This step is important for preparing DNA samples for Ion Torrent Sequencing.

Highlights

Ion Torrent Sequencing, also known as Ion Proton Sequencing, is a fast and reliable DNA sequencing technology from Life Technologies.

The technology's most important advantage is its speed, outperforming Illumina sequencing in terms of reliability.

Ion Torrent Sequencing relies on the release of hydrogen ions during nucleotide sequencing.

The process involves fragmentizing the genome sequence, attaching adapters, and amplifying the DNA fragments.

DNA sequences are attached to insoluble beads, which are then loaded into tiny wells on a microchip.

The microchip used in Ion Torrent Sequencing is analogous to a semiconductor chip with millions of small wells.

Each well serves as a tiny pH meter, capable of detecting the release of hydrogen ions during DNA synthesis.

The sequencing process begins with the addition of a single type of nucleotide at a time to detect the complementary base pairing.

The Ion Sensitive layer detects changes in pH due to the release of hydrogen ions, which is a base call in sequencing.

The technology allows for the sequencing of large genomes in a short amount of time, with a 100 GB chip capable of sequencing billions of DNA fragments.

Ion Torrent Sequencing is significantly faster than other Next Generation Sequencing technologies, taking only 2 to 3 hours for large DNA sequences.

The process does not require fluorescence, resulting in a cleaner and more efficient sequencing method.

The technology scales easily, with chip capabilities increasing from 10 MB to 100 GB, facilitating the sequencing of even larger genomes.

Ion Torrent Sequencing minimizes sequencing time dramatically, providing a significant advantage over other methods.

The sequencing process involves running each nucleotide type sequentially, ensuring accurate base calling.

The technology uses a complex algorithm to align the sequenced fragments and reconstruct the full genome sequence.

Ion Torrent Sequencing is a cost-effective method for rapid and accurate genome sequencing.

Transcripts

welcome back friends welcome to another

video tutorial from shus biology and in

this video tutorial we'll be talking

about ion torrent sequencing which is

also known as ion proton sequencing and

it is from live Technologies it's a new

mode of DNA sequencing technology the

higher end Next Generation DNA

sequencing technology that is incredibly

fast and it is much uh the most

important Advantage is the speed and

also it is very reliable the data that

we get it's very very reliable even

compared with Illumina sequencing that

we know of so let's talk about it so

here will be ion

torrent sequencing okay so what does

this thing mean and what is this ion

torrent sequencing if you look at this

name there is something ions to deal

with this right and ion means here we

are talking about protons that that is

hydrogen

ions now the idea of this DNA sequencing

is just like any other Next Generation

sequencing you know in next Generation

sequencing what we do we have the genome

sequence we fragmentize the sequence

into smaller parts once we fragmentize

the sequence into small fragments then

we put them and attach adapters in both

the ends upon addition of adapters we

amplify those fragment of DNA sequences

and we attach them in beads you know

they different

in insoluble beads are there where we

attach the DNA sequence so once we have

bead filled beads filled with the DNA

sequence then we take each of the

sequence we have a single standard DNA

and then we use uh new deoxy nucleotide

sequences uh to attach one after another

to know the opposite uh sequence of the

Strand which is the actual Target DNA to

be sequenced now same thing will happen

here in case of iron torrent but in this

case it will rely on release of proton

ion release of hydrogen ion every time a

nucleotide sequence is added this is the

chemical point of view of this whole

process you know if you look at a

growing chain of nucleotide sequences if

you look at a growing chain it has a

three prime hydroxy group free to react

and another three phosphate group

containing base will come and what

happens actually this hydroxy group it

will interact and attack the alpha

phosphate so two phosphate groups

released as pyrro phosphate and they

form a bond between between each other

the bond is known as phosphodiester Bond

now once this process is going on this

is the polymerization stage once this

process is going on it will release

proton it will release hydrogen ion

right in ion torrent sequencing we

detect the generation of this hydrogen

ions okay this is the basic chemical

overview

now how this whole process is conducted

now as you know we have the DNA let's

say this is uh say the whole genomic DNA

we talking about it's a big uh part of

the DNA so what we do here we'll

fragmentize this DNA okay

fragmentation so the DNA is fragmented

into small

parts one small part of the DNA is

produced okay double standard DNA all of

them then what will you do

you simply add some adapter sequences at

both the ends why we need adapters

because this adapter

sequence I'm telling you in a moment why

you need them you know right after

adding the adapter we want this DNA

sequences to be added and attached to

beads okay insoluble molecules okay

let's draw the beads

here see this is a bead and what happens

actually our beads containing a specific

type of single standard DNA sequence all

around see single standard DNA sequence

attached to the beads we we synthesize

them and we prepare it in the kit in the

sequencing kit then what we do here in

this case you know the sequence that are

placed in the bead we know right because

we designed it so we prepare the

compliment sequence of this bead as

adapter okay now we make all these DNA

single

stranded okay the the DNA that we

prepare they will be single stranded DNA

okay single strand so now as we know

this section of the adapter is

complimentary to the DNA that is present

in the bead it can easily go and bind

right so if I let you know the binding

it will be like

this isn't

it

by this way many of those sequence will

will go and bind all the sequences they

will go and

bind okay and let me draw all the

compliment DNA sequence that will help

them to

bind right this is how all the

fractioned DNA will bind and attach to

the beads because of this compliment

feature that's why we need to add the

adapter so this is a very common process

for all the type of Next Generation

sequencing so once the bead is produced

now these beads are placed in tiny Wells

okay and the well format that is also

created in the lab is known as microchip

okay it's just like the semiconductor

chip that we find in most of our

electronic appliances today in your

digital camera also you will see small

and small chip okay very very small chip

uh even the dimension of 1 to 2 in uh 1

and 1/2 in uh in the D 1 and 1/2 in

square in this is very small right so

those small chip everything is

accommodated in that chip so what we do

in in in the semiconductor technology

there are small fractions the chips

which are divided into millions and even

billions of small pixels where your

digital camera records the light data

okay once they record the light data

they will conduct the data in form of

binary that is one and zero so that the

camera will understand which part gets

brighter light which part gets uh slower

uh I mean darker light and that's how

the image is constructed now in this

case the same technology but here we

don't rely on the light here instead of

each of the pixels we create that pixel

as whales you know whales whales are

small grooves very tiny grooves and in

this semiconductor cheap in the cheap of

iron torrent sequencing in this case

each of those chap consisting of

millions and billions of small Wells and

each of the wells are recruited for

applying these beads onto them so if I

draw the chip very well if you look at

here like

this and this is

divided and further divided if you take

this and it further divided very tiny

fraction and each of the fractions are

known as Wells now these Wells are

allotted for the beads to present so

beads are accompanying one well each of

the beads are present in one well so we

load it we load those beads into the whs

actually so so those beads are loaded

into the wells and you know not only the

beads but also along with the beads we

have all the DNA sequences also added

coming out of the Beats right just like

this let's see we add these things in

each of the

wells and there are millions of Wells

even billions of Wells containing all

the fractions so you can imagine that a

DNA sequence however long it is you can

fractionate it you can break it down you

can load them into the beads attach them

into the beads and you can put them uh

inside this Wells okay it does not

matter because the wells have enormous

capability of holding DNA uh in attached

with beads so once this thing is done

once we add everything into this Wells

now this is the chip right this chip is

the heart of ion tent sequencing once

the chip is ready once everything is

loaded into the wells this is the loaded

chip now this chip is have uh this chip

has another layer of design and that is

a secondary layer right after the chip

if I draw it as a cross-section view you

will see this is the

chip this is the chip

okay and right next to this chip there

is another

layer this is this is known as

the

Ion Sensitive

layer Ion Sensitive layer which is

created also now this layer can detect

presence of positively charged ion which

is here the protons which is here the

hydrogen ions this is the Ion Sensitive

layer just like your camera right after

the chip there is a sensor CCD sensor or

SOS sensor here we have the ion sens

layer now the idea is we add all these

things we add this chip chip is loaded

now right after the load of Chip we

start adding the nucleotide sequences

one after another okay so everything is

placed this is now the time to run the

sequencing because this is the

preparation phase okay we multiply we

break this DNA down we multiply it uh

even sometimes need amplification uh PCR

amplification to get uh to to take each

of the fragments as much as you you can

to run because you know the more you run

uh this DNA fragments as the whole

sequencing through whole sequencing the

more accurate data you will get okay

because if you run only once to check

the error rate will be higher if you if

you run thousand times and you need to

uh take all the data and need to figure

out what's exactly there it will be more

accurate so here what once after

loading we we start the sequencing now

the sequencing process works like this

okay the sequencing begins with each

nucleotide sequences one nucleotide

sequence at a time we don't know what

exactly the sequence is that's what we

want to do that's what we want to find

out so the idea we know is that there

are four different bases present in the

DNA adenine guanine thyine and cytosine

right four different bases are present

so what we can do we can run this whole

sequencer for each of those nucleotide

sequences one at a time okay so let's

say at the very beginning we start with

only

adenine okay we check for only adenine

so what we do we add DP deoxy adinos

ribon nucleotide triphosphate so once we

add DP wherever there is the

complimentary signal

T this adenine will bind okay and

wherever it is something else it will

not bind so let's say here it's t so the

adenine will go and pair

right ad go pair that's the

complimentary base pairing the easy

stuff always so whenever whenever a

nucleotide is attached right whenever

adenine is attached to the growing chain

here through this adapter the growing

chain at that time it will

release one molecule of hydrogen ion for

each attachment of nucleotide it will

release one molecule of hydrogen ion so

release of one molecule of hydrogen ion

one hydrogen ion is released for a and

that is sensed by the Ion Sensitive

layer they can sense it okay how the

question is how because you know this

whole process is running in all the

wells it does not matter this it's

occurring only at one well let's say

this one but this thing is occurring

every well because we add this adenine

datp in every in this whole chip we load

it in the whole Wells so wherever they

find Adine they will pair it doesn't

matter wherever they find they will pair

so wherever they find they will pair and

after this

pairing they will get the signal in form

of hydrogen ion but here the Ion

Sensitive layer can detect the pH of the

solution because you know hydrogen ion

influence pH so if hydrogen ion is

released the pH of the solution will

change the pH of the chambers will

change right it will drop drop the pH

will go down so here whenever a proper

binding is done hydrogen released

hydrogen ion released and the pH drops

so the change in pH is monitored okay

and the altitude at which the change in

pH is occurring is also monitored okay

now let's say there are consecutive 2 T

consecutive T's are present say 3 T

consecutive three adinin will be

added

so three hydrogen ions will be generated

so the pH change will be

more right and each time the pH change

occurs they count it this this Ion

Sensitive layer count it as a base call

it's known as a base call that is we had

we are checking for base a whenever we

find the pH change that means we get a

base call that means that base is

present definitely so by this way base

call is recorded every time

okay and because this sensitive layer at

the end if you look at the structure of

the

machine it is added ultimately to the to

the CPU the the processing

unit which is getting all the data which

is making everything that so it's it's

ultimately if if you think it it's

ultimately as a pH

meter every of the small tiny Wells are

nothing but tiny PH meters so this chip

is entirely millions and millions of pH

meter combined together they're

functioning together whenever this uh

recognition is occurring base call is

recorded and that data is in uh the CPU

the CPU gets the data it is telling us

yes this base is present now once Three

A's are attached so obviously three T's

are present in the actual stand which we

want to synthesize Now by this way we

can get the data of the whole genome

sequence in each of those fragments then

CPU runs complex algorithm to figure it

out align them together to get the full

data now the major advantage of iron

torrent sequencing is that you know

however big your genome is you can run

it almost once because you know it's the

chip is enormous capability now we have

a chip of 100 GB capability we started

with 10 MB and this capability is

increasing because it's it's the scaling

up of this of this cheap is also very

easy

I mean it's it's also very interesting

so that's why the scaling up is going on

we started with 10 MB now we have 100 GB

capability chip which can run billions

of DNA fragments Al together so what it

does actually it minimize the time

dramatically compared with the other

Next Generation sequencing even compared

with Illumina sequencing in Next

Generation sequencing it will take

almost

30 hours to 48 Hours even for a

sequencers to get a large DNA sequence

uh sequenced but in this case it will

only take two to 3 hours so it's it's

incredibly fast and it's also accurate

because it's running everything at the

same go and as it's running everything

at the same time you you need only one

chip to run that process it is not very

much complex every time you need to do

this stuff and and like not like that it

is uh again compared to that uh though

it's expensive but it's not that much if

you think of uh the cost time that is

providing in 3 hours you can spend that

money uh to sequence the ch

now as I told you for each of the time

it will run for one nucleotide at a time

so once it's run for a then it will wash

off rest of the a once all this process

is done then you'll go for the G and the

same process then you'll go for the T

then wash off same process for the C

wash off this is how the whole thing is

done no fluoresence nothing else

required very clean job and this is very

very important okay so if you like this

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