Ion torrent sequencing
Summary
TLDRThis 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
- π Ion Torrent Sequencing, also known as Ion Proton Sequencing, is a fast and reliable DNA sequencing technology from Life Technologies.
- π¬ The technology is based on the detection of hydrogen ion (proton) release during the polymerization stage of DNA sequencing.
- 𧬠The process involves fragmentizing the genome sequence, attaching adapters, amplifying the fragments, and attaching them to beads.
- π DNA sequences are attached to single-stranded DNA on the beads, which are then loaded into tiny wells on a microchip.
- π‘ The sequencing chip functions like a semiconductor chip with millions of small wells, each capable of holding a bead with a DNA fragment.
- π 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.
- π 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.
- π 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.
- π οΈ The process is straightforward and does not require fluorescence, making it a clean and efficient method for DNA sequencing.
- π The data obtained from the sequencing is highly reliable and can be aligned using complex algorithms to reconstruct the full genome sequence.
- π° 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
π 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.
𧬠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.
π 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.
π 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.
Mindmap
Keywords
π‘Ion Torrent Sequencing
π‘Hydrogen Ion
π‘Phosphodiester Bond
π‘Adapter
π‘Bead
π‘Microchip
π‘Ion Sensitive Layer
π‘Base Call
π‘Sequencing Chip
π‘Deoxyribonucleotide Triphosphate (dNTP)
π‘PCR Amplification
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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