Lecture 08

16 October 2017

Next Generation Sequencing

Ion Semiconductor Sequencing
Illumina synthesis Sequencing
 Ion semiconductor sequencing

Ion semiconductor sequencing is a

method of DNA sequencing based on
the detection of hydrogen ions that
are released during
the polymerization of DNA.

This is a method of "sequencing by

synthesis", during which
a complementary strand is built based
on the sequence of a template strand.
 The Principle

In nature, the incorporation of a deoxyribonucleoside triphosphate (dNTP)

into a growing DNA strand involves the formation of a covalent bond and
the release of pyrophosphate and a positively charged hydrogen ion. A
dNTP will only be incorporated if it is complementary to the leading
unpaired template nucleotide. Ion semiconductor sequencing exploits
these facts by determining if a hydrogen ion is released upon providing a
single species of dNTP to the reaction.
 Steps of Methodology

Uses a semiconductor chip similar to one used in digital camera

The chip (sensing layer) looks like figure ( A ) when light strikes it ; it is
translated into 0s and 1s (digital information) ( B )

An ion chip has millions of wells covered with these pixels in

semiconductor chip.

(A) (B)
 Contd
These wells capture the chemical information from DNA
sequencing and translates them into digital information.

These are the similar beads that were used in

pyro-sequencing. Each bead has attached copies of
millions of same fragment of DNA of interest.

Each bead is deposited in a single

well
 Contd
Again each well is flooded with one
of the nucleotides (out of A,T,C,G)

Whenever a nucleotide is added to

the complementary strand a
hydrogen ion is released.

Actually a pyrophosphate and

hydrogen ion is released; but in
pyrosequencing they made use of
pyrophosphate whereas in this
method they translate this
hydrogen ion into digital output
captured on semiconductor chip.
 Contd
The hydrogen ion changes the pH of the solution in the well.

An ion sensitive layer beneath the well measures this pH change and convert it to
voltage.

So basically each well works as a pH meter.

A pH meter is a scientific instrument that measures the hydrogen-

ion activity in water-based solutions, indicating its acidity or alkalinity expressed
as pH.

The pH meter measures the difference in electrical potential between a pH

electrode and a reference electrode

The process is repeated after every 15 seconds with different nucleotide.

The unattached dNTP molecules are washed out before the next cycle when a
different dNTP species is introduced.
This process happens simulataneously in millions of wells
 Difference from others

This technology differs from

other sequencing technologies in that no
modified nucleotides or optics are used. Ion
semiconductor sequencing may also be
referred to as Ion Torrent sequencing, pH-
mediated sequencing, silicon sequencing, or
semiconductor sequencing.
 Strengths

The major benefits of ion semiconductor sequencing are rapid sequencing

speed and low upfront and operating costs. This has been enabled by the
avoidance of modified nucleotides and optical measurements.

Because the system records natural polymerase-mediated nucleotide

incorporation events, sequencing can occur in real-time. In reality, the
sequencing rate is limited by the cycling of substrate nucleotides through the
system.Ion Torrent Systems Inc., the developer of the technology, claims that
each incorporation measurement takes 4 seconds and each run takes about
one hour, during which 100-200 nucleotides are sequenced. If the
semiconductor chips are improved (work under progress), the number of
reads per chip (and therefore per run) should increase.

The cost of acquiring a pH-mediated sequencer from Ion Torrent Systems Inc.
at time of launch was priced at around $50,000 USD, excluding sample
preparation equipment and a server for data analysis. The cost per run is also
significantly lower than that of alternative automated sequencing methods, at
roughly $1,000.
 Limitations
If homopolymer repeats of the same nucleotide (e.g. TTTTT) are present on
the template strand (strand to be sequenced) then multiple introduced
nucleotides are incorporated and more hydrogen ions are released in a single
cycle. This results in a greater pH change and a proportionally greater electronic
signal. This is a limitation of the system in that it is difficult to enumerate long
repeats. This limitation is shared by other techniques that detect single nucleotide
additions such as pyrosequencing. Signals generated from a high repeat number
are difficult to differentiate from repeats of a similar but different number; e.g.,
homorepeats of length 7 are difficult to differentiate from those of length 8.

Another limitation of this system is the short read length compared to other
sequencing methods such as Sanger sequencing or pyrosequencing. Longer read
lengths are beneficial for de novo genome assembly. Ion Torrent semiconductor
sequencers produce an average read length of approximately 400 nucleotides per
read.

The throughput is currently lower than that of other high-throughput sequencing

technologies, although the developers hope to change this by increasing the
density of the chip.
ILLUMINA SEQUENCING SYNTHESIS
 Steps DNA Bridge Amplification
1) Adapted attached to ends of fragmented DNA
2) Denature and bind to flow cell surface
3) Bridge amplification
4) Denature and repeat to generate clusters os similar
fragments
 Steps Synthesis by termination
Illumina sequencing devices incorporate fluorescent
reversible terminators.
Each dNTP has a corresponding fluorophore attached to it.

When polymerase elongates the strand with a fluorescently-

labeled dNTP, the clusters are then excited by a light source
and the color recorded by an optical detector.

After incorporation
occurs, the
fluorophore is cleaved
 Sample preparation
Genes of interest---as SS fragments
Add adapters to both sides
 Contd
Uses glass slides with two types of oligo
probes which are present all over the slide
and immbolized
 Cluster generation by Solid phase PCR

We add our adapter modified

ssDNA fragments on this glass
slide

One adaptor is complementary to

one of the oligos present on the
glass slide and thus our fragment
becomes attached to it
 Contd
DNA ploymerase completes the
complementary strand

And DNA is denatured and the free DNA

(the original DNA fragment/gene of
interest) is washed away

The new strands are going to be amplified

through illumina created special method
called bridge amplification
DNA bridge amplification
The ssimmbolized newly formed DNA
strand bends over and binds to other
oligo through adaptor sequence added
on other side.
 Contd
DNA polymerase completes the DNA synthesis of these bent fragments
They are denatured
And we have millions/billions of immobilized ssDNA frgaments
The number of cycles determine the length of the read
 Number 1 vendor
Illumina is now the dominant vendor of high-throughput DNA sequencing
machines.

According to the OmicsMaps website, Illumina has 1145 machines in place

worldwide compare to 836 for all other vendors combined!

Since Illumina machines generally produce the highest yields, they are responsible
for an even larger fraction of the total bases of DNA sequence information assayed
worldwide.

The HiSeq produces the highest yields (up to 75 billion bases, or Gb) per sample
with maximum read length of 100 bases from both ends of each template. A
typical HiSeq run processes eight samples in 11 days. The MiSeq is a less
expensive machine that sequences a single sample per run. The MiSeq runs more
quickly (4-24 hours), but produces much less data per sample- from 200 Mb to 2
GB depending on the number of cycles. The MiSeq is capable of reading 150 bp
from each end of a template molecule, but 250 bp reads are currently in beta
testing.
 Next
Solid DNA Sequencing
THANK YOU