Advance sequencing technology

Dr. M.Vinoth,

Animal biotechnology

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INTRODUCTION

DNA sequencing is a fundamental requirement for modern gene manipulation.

The sequencing of the reference human genome was the capstone for many
years of hard work spent developing high-throughput, high capacity production
DNA sequencing and associated sequence finishing pipelines. Despite these
dramatic changes in sequencing and assembly approaches, the primary data
production for most genome sequencing since the HGP has relied on the same
type of capillary sequencing instruments as for the HGP. However,that scenario
is rapidly changing owing to the invention and commercial introduction of
several revolutionary approaches to DNA sequencing, so-called next-generation
sequencing technologies. Although these instruments only began to become
commercially available in 2004, more than 100 next-generation sequencing–
related manuscripts have appeared. These technologies are not only changing
our genome sequencing approaches and the associated timelines and costs,.
Furthermore, next-generation platforms are helping to open entirely new areas
of biological inquiry, including the investigation of ancient genomes, the
characterization of ecological diversity, and the identification of unknown
etiologic agents
OLDER METHODS
1977- maxam–gilbert sequencing
1980-chain-termination methods
NEXT-GENERATION DNA SEQUENCING(NGS)
Three platforms for massively parallel DNA sequencing read production are in
reasonably widespread use at present: the Roche/454 FLX (www.454.com), the
Illumina/Solexa Genome Analyzer (www.illumina.com/), and the Applied
Biosystems SOLiDTM System (www.appliedbiosystems.com).

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Recently, another two massively parallel systems were announced: the Helicos
HeliscopeTM (www.helicosbio.com) and Pacific Biosciences SMRT
(www.pacificbiosciences.com) instruments.

Application
 Discover all types of genetic variation: SNPs, insertions, deletions, copy
number variants, and rearrangements
 Use targeted sequencing of association or linkage peaks to identify
variants that cause disease
 Characterize new bacterial isolates by de novo sequencing and re-
sequencing
 Resequence a collection of samples from any population or species
 Profile DNA methylation status across the entire genome
 Define somatic variations in cancer
 Characterize complex RNA populations for new genes and transcript
structures
 Create new applications enabled by massively parallel sequencing
 Gene expression
 Structural variation analysis
 DNA Sequencing
 Transcriptome Analysis

Roche/454 FLX Pyrosequencer

454 Life Sciences, a Roche company, is a biotechnology company based in

Branford, Connecticut specializing in high-throughput DNA sequencing using a
novel massively parallel sequencing-by-synthesis approach.

In 2005 ,GS20 sequencing machine was relesed,

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In 2008, 454 Sequencing launched the GS FLX Titanium ability to sequence
400-600 million base pairs per run with 400-500 base pair read lengths.

In late 2009, 454 Life Sciences introduced the GS Junior System

Sequencing Chemistry

454 Sequencing, based on sequencing-by-synthesis. This next-generation

sequencer was the first to achieve commercial introduction in 2004 and uses an
alternative sequencing technology known as pyrosequencing.

In pyrosequencing

• Each incorporation of a nucleotide by DNA polymerase results in the

release of pyrophosphate
• Ppi converted into ATP by ATP sulfurylase
• This ATP give energy to luciferase (firefly enzyme)
• Luciferase- oxidize luciferin and generate light
• Before next step apyrase degrade the unincorporated nucleotides
• The amount of light produced is proportional to the number of
nucleotides incorporated.
In the Roche/454 approach

 Nucleotides are flowed sequentially in a fixed order across the PicoTiter

Plate device during a sequencing run.

 During the nucleotide flow, hundreds of thousands of beads each carrying

millions of copies of a unique single-stranded DNA molecule are
sequenced in parallel.

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  If a nucleotide complementary to the template strand is flowed into a
well, the polymerase extends the existing DNA strand by adding
nucelotide(s).

 Addition of one (or more) nucleotide(s) results in a reaction that

generates a light signal that is recorded by the CCD camera in the
instrument.

 The signal strength is proportional to the number of nucleotides

incorporated in a single nucelotide flow.
 This strategy allows the 454 base-calling software to calibrate the light
emitted by a single nucleotide incorporation.

Demerit
However, the calibrated base calling cannot properly interpret long stretches
(>6) of the same nucleotide (homopolymer run), so these areas are prone to
base insertion and deletion errors during base calling.
Each incorporation step is nucleotide specific, substitution errors are
rarely encountered in Roche/454 sequence reads. The FLX instrument currently
provides 100 flows of each nucleotide during an 8-h run, which produces an
average read length of 250 .These raw reads are processed by the 454 analysis
software and then screened by various quality filters to remove poor-quality
sequences.
The resulting reads yield 100 Mb of quality data on
average.,FLXreads are of sufficient length to assemble small genomes such as
bacterial and viral genomes to high quality and contiguity

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Figure 1
 The method used by the Roche/454 sequencer to amplify single-stranded

DNA copies from a fragment library on agarose beads.

 A mixture of DNA fragments with agarose beads containing

complementary oligonucleotides to the adapters at the fragment ends are

mixed in an approximately 1:1 ratio.

 The mixture is encapsulated by vigorous vortexing into aqueous micelles

that contain PCR reactants surrounded by oil, and pipetted into a 96-well

microtiter plate for PCR amplification.

 The resulting beads are decorated with approximately 1 million copies of

the original single-stranded fragment, which provides sufficient signal

strength during the pyrosequencing reaction that follows to detect and

record nucleotide incorporation events. sstDNA, single-stranded template

DNA.

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VisiGen Biotechnologies
VisiGen Biotechnologies, Inc. is developing a revolutionary approach
for DNA sequencing that will enable rapid, inexpensive whole genome
sequencing.

Single-molecule detection, fluorescent molecule chemistry,

computational biochemistry, and genetic engineering of biomolecules,
are combined to create this novel sequencing system.

VisiGen's Sequencing Strategy:

 Engineer both polymerase and nucleotide triphosphates to act

together as direct molecular sensors (nano-sequencing machines)
of DNA base identity.
 Monitor signals from individual nano-sequencing machines
(single molecule detection).
 Acquire data during DNA strand synthesis (real-time detection).
 Create massively parallel arrays (microarrays) of these
nanomachines.
 Achieve a sequencing rate of 1Mb/sec/machine.
 Integrate a system of equipment, software, and consumables for
turn-key laboratory applications.
 Ultimately, sequence an entire human genome for less then $1000.

VisiGen's next generation sequencing technology is distinguished from

other leading developers:

 Exploits the natural process of DNA replication.

 Enhances accuracy and minimally impacts efficiency of DNA
replication.

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  Requires minimal sample amount that is prepared in a non-
destructive way.
 Employs direct analysis and eliminates the need for cloning or
amplification.
 Eliminates sequencing reaction processing and electrophoresis.
 Does not destroy the original template material.
 Does not produce an altered DNA polymer that may negatively
impact polymerase activity

Demerit :cost per run is high

Illumina Genome Analyzer

 The single molecule amplification step for the Illumina Genome Analyzer
starts with an Illumina-specific adapter library, takes place on the oligo-
derivatized surface of a flow cell and is performed by an automated
device called a Cluster Station.
 The flow cell is an 8-channel sealed glass micro fabricated device that
allows bridge amplification of fragments on its surface.
 DNA polymerase can produce multiple DNA copies, or clusters, that
each represent the single molecule that initiated the cluster amplification.
 A separate library can be added to each of the eight channels, or the same
library can be used in all eight, or combinations thereof. Each cluster
contains approximately one million copies of the original fragment,
which is sufficient for reporting incorporated bases at the required signal
intensity for detection during sequencing.
 The Illumina system utilizes sequencing by- synthesis approach in which
all four nucleotides are added simultaneously to the flow cell channels,
along with DNA polymerase, for incorporation into the oligo-primed

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 cluster fragments specifically, the nucleotides carry a base-unique
fluorescent label and the 3_-OH group is chemically blocked such that
each incorporation is a unique event.
 An imaging step follows each base incorporation step, during which each
flow cell lane is imaged in three 100-tile segments by the instrument
optics at a cluster density per tile of 30,000.
 After each imaging step, the 3_ blocking group is chemically removed to
prepare each strand for the next incorporation by DNA polymerase.
 This series of steps continues for a specific number of cycles, as
determined by user-defined instrument settings, which permits discrete
read lengths of 25–35 bases.
 A base-calling algorithm assigns sequences and associated quality values
to each read and a quality checking pipeline evaluates the Illumina data
from each run, removing poor-quality sequences.
Figure 2
The Illumina sequencing-by-synthesis approach. Cluster strands created by
bridge amplification are primed and all four fluorescently labeled, 3_-OH
blocked nucleotides are added to the flow cell with DNA polymerase. The
cluster strands are extended by one nucleotide. Following the incorporation
step, the unused nucleotides and DNA polymerase molecules are washed away,
a scan buffer is added to the flow cell, and the optics system scans each lane of
the flow cell by imaging units called tiles. Once imaging is completed,
chemicals that effect cleavage of the fluorescent labels and the 3_-OH blocking
groups are added to the flow cell, which prepares the cluster strands for another
round of fluorescent nucleotide incorporation.

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Illumina Genome Analyzer

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Applied Biosystems SOLiDTM Sequencer
 The SOLiD platform uses an adapter-ligated fragment library similar to
those of the other next-generation platforms, and uses an emulsion PCR
approach with small magnetic beads to amplify the fragments for
sequencing.
 Unlike the other platforms, SOLiD uses DNA ligase and a unique
approach to sequence the amplified fragments .
 Two flow cells are processed per instrument run, each of which can be
divided to contain different libraries in up to four quadrants.
 Read lengths for SOLiD are user defined between 25–35 bp, and each
sequencing run yields between 2–4 Gb of DNA sequence data.
 Once the reads are base called, have quality values, and low-quality
sequences have been removed, the reads are aligned to a reference
genome to enable a second tier of quality evaluation called two-base
encoding.
Disadvantage
(a) The length of a sequence read from all current nextgeneration platforms is
much shorter than that from a capillary sequencer
(b) Each nextgeneration read type has a unique error model different from that
already established for capillary sequence reads.
Both differences affect how the reads are utilized in bioinformatic analyses,
depending upon the application. For example,in strain-to-reference comparisons
(resequencing), the typical definition of repeat content must be revised in the
context of the shorter read length. In addition, a much higher read coverage or
sampling depth is required for comprehensive resequencing with short reads to
adequately cover the reference sequence at the depth and low gap size needed.

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Figure 3
(a) The ligase-mediated sequencing approach of the Applied Biosystems

SOLiD sequencer. In a manner similar to Roche/454 emulsion PCR

amplification, DNA fragments for SOLiD sequencing are amplified on the

surfaces of 1-μm magnetic beads to provide sufficient signal during the

sequencing reactions, and are then deposited onto a flow cell slide. Ligase-

mediated sequencing begins by annealing a primer to the shared adapter

sequences on each amplified fragment, and then DNA ligase is provided along

with specific fluorescentlabeled 8mers, whose 4th and 5th bases are encoded by

the attached fluorescent group. Each ligation step is followed by fluorescence

detection, after which a regeneration step removes bases from the ligated 8mer

(including the fluorescent group) and concomitantly prepares the extended

primer for another round of ligation.

(b) Principles of two-base encoding. Because each fluorescent group on a

ligated 8mer identifies a two-base combination, the resulting sequence reads can

be screened for base-calling errors versus true polymorphisms versus single

base deletions by aligning the individual reads to a known high-quality

reference sequence.

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Applied Biosystems SOLiDTM Sequencer

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Comparison of Roche, Illumina, and Applied Biosystems
NGS Platforms.

Roche (454): Illumina: Applied

Titanium series Genome Biosystems:
reagents (run Analyser II SOLiDTM
on FLX
Genome
Sequencer)
Sequencing pyrosequencin polymerase- Ligation-
chemistry g based based
sequencing- sequencing
by-synthesis
Amplificatio Emulsion PCR Bridge Emulsion
n approach amplification PCR
MB/run 400-600 MB 1300 MB 3000 MB
Time/run 10 hr 4 days 5 days
Read length 400 bp up to 75 bp 35 bp
cost Higher other low low
two

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HeliScope™ Single Molecule Sequencer
Principle:
Within two flow cells, billions of single molecules of sample DNA are captured
on an application-specific proprietary surface. These captured strands serve as
templates for the sequencing-by-synthesis process:
 Polymerase and one fluorescently labeled nucleotide (C, G, A or T) are
added.
 The polymerase catalyzes the sequence-specific incorporation of
fluorescent nucleotides into nascent complementary strands on all the
templates.
 After a wash step, which removes all free nucleotides, the incorporated
nucleotides are imaged and their positions recorded.
 The fluorescent group is removed in a highly efficient cleavage process,
leaving behind the incorporated nucleotide.
 The process continues through each of the other three bases.
 Multiple four-base cycles result in complementary strands greater than 25
bases in length synthesized on billions of templates—providing a greater
than 25-base read from each of those individual templates.

The HeliScope™ Single Molecule Sequencer is the first genetic analyzer to

harness the power of direct DNA measurement, enabled by Helicos True Single
Molecule Sequencing (tSMS)™ technology.  As the world’s first DNA
microscope, the HeliScope instrument performs tSMS chemistry and captures

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images to observe sequencing-by-synthesis reactions for billions of individual
DNA molecules in parallel.

Advantage :
 Detects incorporation of individual bases on individual strands,
eliminating the need for amplification and its associated biases and errors.
 Produces “digital data”, avoiding errors caused by de-phasing or intensity
averaging techniques, and thereby generates consistently low error rates
across all read lengths, resulting in high quality experiments.
 Offers simple sample preparation and high sample capacity per run,
 A throughput performance almost 100X greater than Sanger methods, and
faster than any of the "next-generation" methodologies.  

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 A schematic of the sequencing-by-
synthesis approach used by Helicos
Biosciences. (Courtesy of Helicos
Biosciences.)

Single Molecule Real Time (SMRT™) DNA Sequencing

Instead of inspecting DNA copies after polymerase has done its work, SMRT
sequencing watches the enzyme in real time as it races along and copies an
individual strand stuck to the bottom of a tiny well. Every nucleotide used to
make the copy is attached to its own fluorescent molecule that lights up when
the nucleotide is incorporated. This light is spotted by a detector that identifies
the color and the nucleotide -- A, C, G, or T. By repeating this process
simultaneously in many wells, the technology hopes to bring about a substantial
boost in sequencing speed.

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Advantage
 The SMRT Cell, which enables single molecule, real-time observation of
individual fluorophores against a dense background of labeled nucleotides while
maintaining a high signal-to-noise ratio.
 Phospholinked nucleotides, which enable long readlengths by producing a
completely natural DNA strand through fast, accurate, and processive DNA
synthesis, and
 A novel detection platform that enables single molecule, real-time detection
as well as flexibility in run configurations and applications

Pacific Biosciences has a Single Molecule Real-Time (SMRT) DNA

sequencing, due to be released commercially in 2010 and could enable $100
genome sequencing in 15 minutes in 2013. The second generation real time
DNA reader in 2013 is the one that is expected to hit the $100 genome
sequencing price. They will release a product in 2010 but it will not be that
cheap.

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Both excitation and detection occur without interruption through the transparent
glass bottom of the SMRT Cell.

Enzymatic incorporation of the labeled nucleotide creates a flash of light, which

is converted into a base call using optimized algorithms

CONCLUSION

Genome sequencing will change the practice of medicine from treating to

preventing disease. The shift in patient care from disease treatment to disease
prevention decreases the cost of health care dramatically. “Though in its early
stages, sequencing provides a realistic hope for a new and better approach to
health care in the coming century.”

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Reference
 http://www.454.com/enablingtechnology/the-system.asp

 http://www.illumina.com/pages.ilmn?ID=203

 http://marketing.appliedbiosystems.com

 www.helicosbio.com

 www.pacificbiosciences.com

 http://www.dnamicroarray.com/products_stemcells.html

 Arjournals.annualreviews.org

 VisiGen Biotechnologies Inc. - Technology Overview.htm

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