on the Sanger instrument i can get up to 800 base pair fragments read and given that the size of my
genome is 3 billion base pairs i would need... what? 100 years? i don't have 100 years there
has to be a better way surely! we're looking at how to sequence dna but this time on the illumina
platform illumina is a market dominating company for dna sequencing and so we're looking at their
platform which will be representative of the next generation sequencing platforms so a quick
outline to sequence dna you're basically going to extract the dna from the source organism so if
you're looking at animals you're going to extract the dna from the blood from cells or from tissues
then you're going to fragment the dna you're then going to anneal adapters and index and then you're
going to introduce it to a cell a flow cell which is a glass slide essentially where amplification
will occur and then it's moved into a part of the machine that will conduct the dna sequencing so
let's get a bit of more of an introduction dna is the molecule that carries your genetic
instructions for development function growth and reproduction for all living organisms even some
viruses there are different types of cells in the body you must know that they all contain the same
genome the differences that we see in the cells shape and function like cells in the
lungs function differently from cells in your eye the differences really are just due to what type
of genes are expressed and which ones are not in those cells in those tissues in those organs so
they contain the same genome they contain the same genetic material but depends on what's expressed
and what is not a human genome is three gigabases three gigabases or three billion base pairs
in size so if all the dna in itself were to be pulled out and stretched it would be about 2
meters in length yet they are packaged into cells that are only about 10 microns across
there's some neat folding that has to go on for this to happen to achieve this dna is folded in a
very methodical way the characteristic chromosome arrangements that you may have come across is a
clear example so usually when you look at what is called a karyotype you see all the chromosomes
of the organism and so this is an example of how dna can be folded and arranged interesting
you only see this kind of arrangement during cell division most of the time the dna is not arranged
in this way it's a lot more relaxed so that the information in them can be accessed humans have 22
pairs of chromosomes that are called autosomes so they don't contain any information that determines
whether you're a boy or a girl so you get one of these from each parent so in addition to your 22
pairs of autosomes you also have one pair that is called a sex chromosome and that's because
that determines whether the individual is male or female i mentioned that the size of the human
genome size is three gigabases which is the same as three billion base space three billion base
space i mean think of three thousand base pairs that's a long string of information what about
three million and so this is three billion they we don't have technology that will allow you to just
continue sequencing dna for three billion base pairs right so to in order to sequence
dna you actually have to have it um chopped up into smaller pieces in a gentle way in a
specific controlled way you want to fragment it the term is fragment fragment the dna into
sections that will allow it to fit the particular platform that you want to sequence on so we we are
looking at the illumina platform in this instance and the illumina platform you need between 300 to
600 base pairs it's able to do 300 to 600 base pairs so the aim is to get the dna fragmented or
chopped up into these pieces so that you can go ahead and use the instrument so that it matches
the requirements of the instrument for you to be able to sequence the dna to see what base
pairs what adenines thymine cytosines guanines make up those genetic that genetic material.
in the previous video we looked at how Sanger DNA sequencing works the Sanger approach is called a
traditional sequencing because it was the first technique for sequencing it was the technique
that was used in the human genome project back in the 90s and the human genome project took
actually 13 years to complete 90 percent of the three billion base pairs the entire human
genome actually took a lot longer so that when the announcement was made uh 13 years into the project
it was only 90 percent that was completed the entire human genome the script became available
after 20 years of sequencing this was due really to the amount of information that
had to be processed that had to go through using the sanger sequencing method meant that you could
sequence fragments up to 700 base pairs that's the longest you could get and you could run multiple
so you could have 384 pieces at any one time that would go into the instrument and so by the end of
the day you run it constantly if you have 700 base pairs max that you can produce and you've got 284
you know you kind of repeat the process you would have about a million basis of sequence so this
takes time to get through three billion base pairs of sequence and usually you can't just sequence a
genome once and be like oh yeah i know where every bass plays no things happen some signals are faint
some signals are you know multiple signals come through so the peaks are not so sharp it's not
so distinct so on average you have to actually cover your genome like seven times to really
be confident that each call that you're making each nucleotide that you're saying
is the proper order is actually where it is so if you were to use the human genome
project as an example and try and sequence that on a sangha instrument and have that seven times
what is referred to as coverage the seven times that you kind of have to sequence it
the seven times coverage it would actually take you a hundred years to sequence an entire human
genome obviously the human genome project did not take a hundred years that's because there
were multiple Sanger instruments running day in and day out but you do we still knew that
we needed something a little bit faster than the Sanger capacity so that's what brings us to
what is called the next generation of sequences or next generation sequencing and the flagship the
dominant player in this field is a company called Illumina and so we're gonna get deep into how
you process samples for sequencing on an illumina platform the illumina platform became available in
the 2000s now these systems allow you to sequence thousands to millions of dna molecules at the same
time so it's often referred to as massively parallel sequencing because you're sequencing
thousands to millions of dna molecules all in one reaction so let's take a look at how to
prepare and sequence dna on a next generation DNA sequencer such as the illumina platform so
the first thing is to carefully fragment the dna that you intend to sequence after extracting it
you can do this fragmentation via enzyme specific enzymes that will cut particular nucleotides
or you can use physical sources of fragmenting shearing and the physical forces usually use sound
to fragment the dna and there's a little bit more work to be done on it after fragmenting
the dna for the illumina platform and other next generation sequencing platform
you go ahead and anneal on or like it on some adapters these adapters are unique in that they
have capture sequences and they will come in handy in your next steps but they also have
primal binding sites so they have a forward primer and a reverse primer already as part of the
adapter and this is what the polymerase is going to start to initiate synthesis of a new nucleotide
dna is replicated in a semi-conservative manner in that you always start off with an existing
template and then the enzyme the polymerase copies what's there and replicates that and that's how
you're able to tell what was there in the first place because the polymerase will just match what
was existing and so you know that adenines pair with thymine guanines pair with cytosine and so
acid acid you're like oh so it was an adenine oh it was a thymine oh it was like one in oh it was
a cytosine whatever the order is and of course the primer orientates you as to which order the
sequencing is happening now the next step is where the capture sequences are going to be handy so you
go ahead and you denature the dna dna when you heat up the temperature the strands will separate
and when this happens you can then introduce the strand to the flow cell so the flow cell is like
this microscope slide they already have short dna pieces where oligonucleotides they are called so
nucleotides make up the dna sequences so they've already got these nucleotides already on the
surfaces oligonucleotides and so your capture sequences will be able to complement that and
match to it so there's a flow cell the illumina flow cell the h channels and they have different
sizes which equates to different capacities so different amounts of data that you can get out of
it and so in your flow cell your capture sequences will anneal to them and then the first step for
you to be able to get a strong enough signal that you can be confident with that the detector
in the machine can make their calls based on is to go ahead and amplify so each annealed
dna fragment to the flow cell there will be several cycles of pcr which will result
in at least 1000 clusters for each fragment so that would generate enough of a signal for the
detector to know whether it was an abdomen it will actually form a bridge once you've copied once the
polymerase has copied the strand you end up with a copy of your forward strand and your reverse
strand and the process will be repeated in step six until you have about a thousand copies of each
fragment on the illumina platform taking a look at the four channel chemistry this happens to be
a scenario where each of the four nucleotides have a unique fluorescent marker a fluorescent signal
so what happens is as at each cycle of sequencing all the four nucleotides are released and if they
are the correct match for the templates where the temp the polymerase is elongating the template
then it will incorporate and then the machine will pause and take four images corresponding to
the four wavelengths of the fluorescent per the fluorescent markers on each of the nucleotides
and so by assessing the four images at each cycle and seeing where the signal actually is you'll
be able to tell whether there was an adenine thymine cytosine or guanine that was incorporated
in addition each nucleotide fluorescently labeled nucleotide has a terminator so
when it gets incorporated the elongation process by the polymerase will terminate
and this gives the instrument enough time to image the incorporated nucleotide it's only after it's
done this that it will flow reagents that will remove the terminator so that the next nucleotide
can be floating and the correct nucleotide will get incorporated as per the dna template
so when it's incorporated it emits light and then the instrument is essentially like
really powerful microscopes that will be able to detect the light that's being emitted by each of
the clusters that you've amplified and as you know the dna sequencing will happen with the
phosphates of the new sequence being joined to the hydroxyl in the three prime position of the
previous nucleotide as each incorporation happens you get a different color signal depending on the
base that is incorporated that's how sequencing happens on next generation systems in addition to
your different colored signal which you know is quite similar to the Sangar you have the option
on the next generation systems of having your sequencing runs being multiplexed so you can have
dna from multiple sources multiple experimental conditions and you can have unique indices
indexes that you can ligate onto the adapters as in addition to the adapters so that you'll
be able to then at the next step at the data analysis side separate which sequences came from
which experimental condition or which organism if you want to collect different organisms or which
genome you'll be able to separate with genome if you're multiplexed triplex just means you ran
an experiment that had genomes from different sources or different conditions so before we
end let's have a look at the data so the data that comes out the output is called a bcl file
bcl stands for binary base call so that's the output that you get a binary base call
and where you had multiplexing happening which is quite common you can separate out the samples
based on the index that you would have put onto each of the samples each of the unique samples
and so it's quite typical to go ahead and separate out based on the index and once you transfer
once you do that process the file format that you get is actually called a fast queue file and the
fast queue is is a text based format for storing the nucleotide sequences and it also gives you
an associated quality score the quality score is important because it tells you how reliable
that read is that base call how reliable it is in some instances like i mentioned you may have
i mentioned earlier on you may have had a situation where the signal was really faint
and the instrument made a call but maybe it wasn't that confident about that chord so it gives you a
quality score or maybe it had a long string of the same nucleotide and it kind of the signal became a
little bit confused in that instance so it wasn't that confident of that chord you you each read
each base call each nucleotide call will have an associated confidence score and that's what the
what information the fastq file will give you the bcl file will just be a binary format you wouldn't
be able to know what the quality score is but when the indexes are separated you will be able to have
the quality score associated with that in the and that would be a form called a fast queue format so
most researchers would want the fast queue rather than the bcl file so let's do a summary next
generation sequencing just like the traditional sequencing you would extract dna from blood
cells tissues and just like the traditional you would fragment the dna but unlike the traditional
senior sequencing you would be able to anneal adapters and index in the processing step you
would then introduce the dna that the fragments that you've made the dna library into a flow cell
and in the flow cell amplification will occur so that the detector will have a strong enough signal
and then you will go ahead and sequence the dna you usually have fluorescence as an indicator of
which nucleotide is being incorporated which is similar to the sanger as well
that's it for an example of a next generation sequencing thank you for watching go ahead and
hit the subscribe button to join the study group i will catch you in the next video god bless you