Illumina infinium sequencing

Illumina Infinium Sequencing
Introduction, Process & Advantages
Presented by: AYUSH JAIN
PALB 7286
(Jr. M.Sc.) plant biotechnology

WHAT IS DNA SEQUENCING??
• It is the process of determining the precise order of nucleotides
bases within a DNA molecule
• The canonical structure of DNA has four
bases: thymine (T), adenine (A), cytosine (C), and guanine (G).
DNA sequencing is the determination of the physical order of
these bases in a molecule of DNA.
• The foundation for sequencing was first laid by the work of Fred
Sanger who by 1955 had completed the sequence of all the amino
acids in insulin, a small protein secreted by the pancreas.
• The first DNA sequences were obtained in the early 1970s by
academic researchers using laborious methods based on two-
dimensional chromatography.
Ayush Jain,PBT 503(Molecular Cell Biology & Molecular Genetics) UAS, Bangalore 2
Two-dimensional chromatography is a type of
chromatographic technique in which the
injected sample is separated by passing
through two different separation stages.

GENRATIONS OF DNA SEQUENCING
• The first method for determining DNA sequences
was a location-specific primer extension strategy
confirmed by Ray Wu at Cornell University in
1970.
• Frederick Sanger then modified this primer-
extension strategy to develop more rapid DNA
sequencing methods at the MRC
Centre, Cambridge, UK and published a method
for "DNA sequencing with chain-terminating
inhibitors" in 1977.
• Walter Gilbert and Allan Maxam at Harvard also
developed sequencing methods, including one
for "DNA sequencing by chemical degradation"

TERMINATING CHAIN METHOD
(SANGER SEQUENCING)
• Sanger sequencing is a “first-generation” DNA sequencing method.
• In Sanger sequencing, the target DNA is copied many times, making fragments
of different lengths. Fluorescent “chain terminator” nucleotides mark the ends of
the fragments and allow the sequence to be determined.
• In the Human Genome Project, Sanger sequencing was used to determine the
sequences of many relatively small fragments (900 bp or less) of human DNA.
• Sanger sequencing gives high-quality sequence for relatively long stretches of
DNA (up to about 900bp). It's typically used to sequence individual pieces of
DNA, such as bacterial plasmids or DNA copied in PCR.
• Sanger sequencing is expensive and inefficient for larger-scale projects, such as
the sequencing of an entire genome or metagenome (the “collective genome” of a
microbial community).

ingredients of sanger sequencing
A DNA polymerase
enzyme
A primer, which is a
short piece of single-
stranded DNA that
binds to the template
DNA and acts as a
"starter" for the
polymerase
The four DNA
nucleotides (dATP,
dTTP, dCTP, dGTP)
The template DNA to
be sequenced
•Dideoxy, or chain-
terminating, versions
of all four nucleotides
(ddATP, ddTTP,
ddCTP, ddGTP), each
labeled with a
different color of dye

DNA to be sequenced
is denatured
A sequencing
primer is annealed
to the single
stranded DNA
DNA polymerase
extends the primer in
5-3 direction which
can be randomly
terminate by ddNTP’s
4 separate reactions
are prepared with
small amount of one
of the 4 ddNTP’s and
all dNTP’s producing
4 separate set of
classes
Heat this partially double
stranded molecule and
add denaturating agent
(formaldehyde) & SS
termination molecule are
released from template
Molecules are separated
using high resolution gel
electrophoresis
The sequence of the original
region of DNA is then finally
deduced by examining the
relative positions of the
dideoxynucleotide chain
termination products in the four
lanes of the denaturing gel

Maxam-gilbert chemical chain termination
method

Short-gun DNA sequencing

Next generation sequencing (ngs)
• The most recent set of DNA sequencing technologies are collectively referred to
as next-generation sequencing.
• Most of the next-generation sequencing techniques share a common set of features
that distinguish them from Sanger sequencing.
a) Highly parallel: many sequencing reactions take place at the same time.
b) Micro scale: reactions are tiny and many can be done at once on a chip.
c) Fast: because reactions are done in parallel, results are ready much faster.
d) Low-cost: sequencing a genome is cheaper than with Sanger sequencing.
e) Shorter length: reads typically range from 50-700 nucleotides in length.

Basic steps in all ngs methods
LIBRARY
PREPARATION
Random fragmentation
of DNA
Adaptors are then
ligated to these
fragments
AMPLIFICATION
Emulsion PCR
Bridge PCR
SEQUENCING
Sequencing by
synthesis
Pyrosequencing
Ion semiconductor
sequencing
Sequencing by
ligation
Sequencing by ligation
Bioinformatical
analysis

STEP-1 LIBRARY PREPRATION
DNA is fragmented either
enzymatically or by
sonication (excitation using
ultrasound) to create smaller
strands
Adaptors are short,
double-stranded pieces of
synthetic DNA that are then
ligated to these fragments
with the help of DNA ligase,
an enzyme that joins DNA
strands. adaptors enable the
sequence to become bound
to a complementary
counterpart.
Library fragments need to be
spatially clustered in PCR
colonies or 'polonies' as they
are conventionally known,
which consist of many copies
of a particular library
fragment.

Step-2 amplification
Library amplification is
required so that the
received signal from the
sequencer is strong
enough to be detected
accurately.
In Enzymatic
amplification, phenomena
such as 'biasing' and
'duplication' can occur
leading to preferential
amplification of certain
library fragments as in
sanger and Maxam–gilbert
sequencing. So we use
Emulsion PCR Bridge PCR

Emulsion pcr
1.Emulsion oil,
beads, PCR mix
and the library
DNA are mixed
to form an
emulsion which
leads to the
formation of
micro wells
2.The PCR then
denatures the
library fragment
leading two
separate strands,
one of which (the
reverse strand)
anneals to the
bead
3.The annealed
DNA is
amplified by
polymerase
starting from the
bead towards
the primer site.
4.The original
reverse strand
then denatures
and is released
from the bead
only to re-
anneal to the
bead to give two
separate strands.
5. These are
both amplified
to give two
DNA strands
attached to the
bead
6.The process
is then
repeated over
30-60 cycles
leading to
clusters of
DNA.

Bridge pcr
1.The surface of the
flow cell is densely
coated with primers
that are
complementary to
the primers attached
to the DNA library
fragments
2.The DNA is then
attached to the
surface of the cell at
random where it is
exposed to reagents
for polymerase
based extension
3.On addition of
nucleotides and
enzymes, the free
ends of the single
strands of DNA
attach themselves to
the surface of the cell
via complementary
primers, creating
bridged structures
4.Enzymes then interact
with the bridges to
make them double
stranded, so that when
the denaturation occurs,
two single stranded
DNA fragments are
attached to the surface
in close proximity
5. Repetition of
this process leads
to clonal clusters
of localized
identical strands.

Step-3 sequencing
Several competing methods of Next Generation Sequencing have been developed
a) 454 Pyrosequencing
• Pyrosequencing detects the release of pyrophosphate(phosphorus oxyanion)
when nucleotides are added to the DNA chain.
• It uses the emulsion PCR technique to construct the polonies required for
sequencing and removes the complementary strand.
• The incorporation of correct dNTP enzymatically into the strand releases
• In the presence of ATP sulfurylase and adenosine, the pyrophosphate is converted
into ATP. This ATP molecule is used for luciferase-catalyzed conversion of luciferin
to oxyluciferin, which produces light that can be detected with a camera.
• The relative intensity of light is proportional to the amount of base added (i.e. a
peak of twice the intensity indicates two identical bases have been added in
succession)

b) Sequencing by ligation (SOLiD)
• SOLiD is an enzymatic method of sequencing that uses DNA ligase to ligate double-stranded DNA
strands
• Emulsion PCR is used to immobilize/amplify a ssDNA primer-binding region which has been
conjugated to the target sequence on a bead.
• These beads are then deposited onto a glass surface
• Once bead deposition has occurred, a primer of length N is hybridized to the adapter, then the beads
are exposed to a library of 8-mer probes which have different fluorescent dye at the 5' end and a
hydroxyl group at the 3' end.
• Only a complementary probe will hybridize to the target sequence, adjacent to the primer
• A phosphorothioate linkage between bases 5 and 6 allows the fluorescent dye to be cleaved from the
fragment using silver ions. This cleavage allows fluorescence to be measured (four different
fluorescent dyes are used, all of which have different emission spectra) and also generates a 5’-
phosphate group which can undergo further ligation
• Once the first round of sequencing is completed, the extension product is melted off and
then a second round of sequencing is performed with a primer of length N−1. Many rounds
of sequencing using shorter primers each time (i.e. N−2, N−3 etc.) and measuring the
fluorescence ensures that the target is sequenced.

ADVANTAGES
• Due to the two-base sequencing
method (since each base is effectively
sequenced twice), the SOLiD
technique is highly accurate (at
99.999% with a sixth primer, it is the
most accurate of the second
generation platforms)
• The SOLiD technique is inexpensive
• It can complete a single run in 7 days
and in that time can produce 30 Gb of
data
• Unfortunately, its main disadvantage
is that read lengths are short, making
it unsuitable for many applications.

Sequence by synthesis
(illumina Infinium)
• It was developed by Shankar Balasubramanian and David Klenerman of
Cambridge University, who subsequently founded Solexa , a company later
acquired by Illumina
• This sequencing method is based on reversible dye-terminators that enable the
identification of single bases as they are introduced into DNA strands
• Consist of 4 basic as any of NGS methods, which are
a) Step 1 sample prep
b) Step 2 Cluster generation
c) Step 3 Sequencing
d) Step 4 Data analysis

Step 1:
• The process begins with purified
DNA.
• After the purification of DNA
Enzymes called transposomes
randomly cut the DNA into short
segments (“tags”) called the
tagmentation step.
• Adapters are added on either side of
the cut points (ligation). Strands that
fail to have adapters ligated are
washed away.
• The next step is called reduced cycle
amplification. During this step,
sequences for primer binding, indices,
and terminal sequences are added.
• Indices are usually six base pairs long
and are used during DNA sequence
analysis to identify samples.

• During analysis, the computer will group all reads
with the same index together.
• The terminal sequences are used for attaching the
DNA strand to the flow cell
• This process takes place inside of an acrylamide-
coated glass flow cell
• The flow cell has oligonucleotides (short nucleotide
sequences) coating the bottom of the cell, and they
serve to hold the DNA strands in place during
sequencing
• The oligos match the two kinds of terminal sequences
added to the DNA during reduced cycle amplification
• As the DNA enters the flow cell, one of the adapters
attaches to a complementary oligo.

Step 2:
• Clusters are generated through bridge
amplification.
• The goal is to create hundreds of identical
strands of DNA. Some will be the forward
strand; the rest, the reverse.
• Polymerases move along a strand of DNA,
creating its complementary strand.
• The original strand is washed away, leaving
only the reverse strand.
• At the top of the reverse strand there is an
adapter sequence
• The DNA strand bends and attaches to the
oligo that is complementary to the top adapter
sequence

• Polymerases attach to the reverse strand, and its
complementary strand (which is identical to the
original) is made.
• The now double stranded DNA is denatured so
that each strand can separately attach to an
oligonucleotide sequence anchored to the flow
cell.
• One will be the reverse strand; the other, the
forward. This process is called bridge
amplification, and it happens for thousands of
clusters all over the flow cell at once.
• Over and over again, DNA strands will bend and
attach to oligos. Polymerases will synthesize a
new strand to create a double stranded segment,
and that will be denatured so that all of the DNA
strands in one area are from a single source
(clonal amplification)

Step 3:
• At the end of clonal amplification, all of the reverse
strands are washed off the flow cell, leaving only
forward strands.
• Primers attach to the forward strands and add
fluorescently tagged nucleotides to the DNA
strand. Only one base is added per round
• Using the four-colour chemistry, each of the four
bases has a unique emission, and after each round,
the machine records which base was added.
• Nucleotides are distinguished by either one of two
colours (red or green), no colour ("black") or
binding both colours (appearing orange as a
mixture between red and green).
• Once the DNA strand has been read, the strand that
was just added is washed away.

• Then, the index 1 primer attaches, polymerizes the index 1 sequence, and is
washed away.
• The strand forms a bridge again, and the 3’ end of the DNA strand attaches to an
oligo on the flow cell. The index 2 primer attaches, polymerizes the sequence,
and is washed away.
• A polymerase sequences the complementary strand on top of the arched strand
• They separate, and the 3’ end of each strand is blocked. The forward strand is
washed away, and the process of sequence by synthesis repeats for the reverse
strand.

Step-4 bioinformatical analysis
• The sequencing occurs for millions of clusters at once, and each
cluster has ~1,000 identical copies of a DNA insert.
• The sequence data is analyzed by finding fragments with
overlapping areas, called contigs, and lining them up.
• If a reference sequence is known, the contigs are then compared to
it for variant identification.
• Illumina read lengths are not very long (Hi-Seq sequencing can
produce read lengths around 90 bp long ) which makes it difficult
to resolve short tandem repeat areas.
• Also, if the sequence is de novo and so a reference doesn’t exist,
repeated areas can cause a lot of difficulty in sequence assembly.

Ayush Jain,PBT 503(Molecular Cell Biology & Molecular
Genetics) UAS, Bangalore
31

ADVANTAGES OVER OTHER METHODS
– Cheaper then pyrosequencing and sanger sequences. HiSeq X® Ten System, released in
2014, can sequence over 45 human genomes in a single day for approximately $1000 each.
– Due to automated nature of Illumina sequencing it is possible to sequence multiple
strands at once and gain actual sequencing data quickly ( due to paired end sequencing).
– Library preparation time have reduced from 1-2 days from early NGS method to 90 min
in illumine sequencing.
– Because the dynamic range with illumina is adjustable and nearly unlimited, researchers
can quantify subtle gene expression changes with much greater sensitivity than
traditional methods
– The sample throughput per run in NGS has also increased over time. Multiplexing allows
large numbers of libraries to be pooled and sequenced simultaneously during a single
sequencing run.
– NGS technology is also highly flexible and scalable. Illumina NGS instruments range
from the benchtop MiniSeq™ System, with output ranging from 1.8–7.5 Gb for targeted
sequencing studies, to the NovaSeq™ 6000 System, which can generate an impressive 6
Tb and 20 B reads in ~ 2 days† for population-scale studies.

QUESTIONS ???

BIBLIOGRAPHY
1. Web References
• www.illumina.com
• http://dnatech.genomecenter.ucdavis.edu
• www.khanacademy.org
• en.wikipedia.org
• http://www.atdbio.com
2.Books
• Biotechnology by u. satayanayrana
• Genome 3 by T.A.Brown

3. Video references
• YouTube channel – shomu’s biology
• You Tube channel- illumina.com
4. Research papers
• Journal genomics (21 NOV 2008) Title: Generations of sequencing technologies
Erik Pettersson , Joakim Lundeberg, Afshin Ahmadian Department of Gene
Technology, Royal Institute of Technology (KTH), AlbaNova University Center,
Roslagstullsbacken 21, SE - 106 91 Stockholm, Sweden.
• Journal BMC Bioinformatics (2012) Title: A comparison of feature selection and
classification methods in DNA methylation studies using the Illumina Infinium
platform Joanna Zhuang1,2, Martin Widschwendter2 and Andrew E
Teschendorff1.
• Journal New Biotechnology Volume 25, Number 4 April 2009 Title: Next-
generation DNA sequencing techniques Wilhelm J. Ansorge Ecole Polytechnique
Federal Lausanne, EPFL, Switzerland

TThankyo
u

Illumina infinium sequencing

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