NEXT GENERATION SEQUENCING

Name

: Alagar . S

Reg no

: 2014419002

Branch

: M.Tech / Computational Biology

Subject code : BT8001

Subject

: Advanced Technologies in Omics Sciences

DNA SEQUENCING
DNA

sequencing is

the

process

of

determining

the

precise

order

of nucleotides within a DNA molecule. It includes any method or technology

that is used to determine the order of the four bases adenine, guanine, cytosine,
and thymine in a strand of DNA. The advent of rapid DNA sequencing methods
has greatly accelerated biological and medical research and discovery.

NEXT GENERATION SEQUENCING

Next-generation sequencing refers to non-Sanger-based high-throughput DNA
sequencing technologies. Millions or billions of DNA strands can be sequenced
in parallel, yielding substantially more throughput and minimizing the need for
the fragment-cloning methods that are often used in Sanger sequencing of
genomes.
ABOUT PACIFIC BIOSCIENCES
Pacific Biosciences is a biotechnology company founded in 2004 that develops
and manufactures systems for gene sequencing. They use single molecule real
time sequencing (SMRT) as their platform, based on the properties of zeromode waveguides. The companys first scientific instrument, called the PacBio
RS. A new version of the sequencer called the "PacBio RS II" was then
released. It produces longer sequence reads and offers higher throughput than
the original instrument. In October of 2014 the company released an updated
chemistry with a mean read length of 10,000 to 15,000 bases and longest reads
exceeding 40,000 bases.

ZERO MODE WAVEGUIDE

A zero-mode waveguide was developed by Pacific Biosciences, is an optical

waveguide that guides light energy into a volume that is small in all dimensions
compared to the wavelength of the light. The zero-mode waveguide (ZMW) is a
nanophotonic confinement structure that consists of a circular hole in an
aluminum cladding film deposited on a clear silica substrate.[3] The ZMW holes
are ~70 nm in diameter and ~100 nm in depth. Due to the behaviour of light
when it travels through a small aperture, the optical field decays exponentially
inside the chamber.[4] The observation volume within an illuminated ZMW is
~20 zeptoliters (20 X 10 21 liters). Within this volume, the activity of DNA
polymerase incorporating a single nucleotide can be readily detected.

SINGLE MOLECULE REAL TIME SEQUENCING (SMRT)

Single Molecule Real Time Sequencing is the principle technology developed

and used for DNA sequencing by pacific biosciences. Single molecule real time

sequencing

utilizes

the zero-mode

waveguide.

single

DNA

polymerase enzyme is affixed at the bottom of a ZMW with a single molecule

of DNA as a template. The ZMW is a structure that creates an illuminated
observation volume that is small enough to observe only a single nucleotide of
DNA (also known as a base) being incorporated by DNA polymerase. Each of
the four DNA bases is attached to one of four different fluorescent dyes. When
a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is
cleaved off and diffuses out of the observation area of the ZMW where its
fluorescence is no longer observable. A detector detects the fluorescent signal of
the nucleotide incorporation, and the base call is made according to the
corresponding fluorescence of the dye.
SMRT technology mimics the natural process of DNA replication, which is a
highly efficient and accurate process. The enzyme responsible for replicating
DNA in nature is called the DNA polymerase. The DNA polymerase attaches
itself to a strand of DNA to be replicated, examines the individual base at the
point it is attached, and then determines which of four building blocks, or
nucleotides, is required to replicate that individual base. After determining
which nucleotide is required, the polymerase incorporates that nucleotide into
the growing strand that is being produced. After incorporation, the enzyme
advances to the next base to be replicated and the process is repeated. SMRT
technology enables the observation of DNA synthesis as it occurs in real time.
To overcome the challenges inherent in observing an enzyme that is 15
nanometers, or nm, in diameter running in real time, the following key
components are used

The SMRT Cell

Phospholinked nucleotides
The PacBio RS II

SMRT CELL

SMRT DNA sequencing is performed on SMRT Cells, each patterned with

150,000 zero-mode waveguides or ZMWs. Single polymerases are immobilized
in the ZMWs, which provide the windows to observe DNA sequencing in realtime. The PacBio RS System continuously monitors 150,000 ZMWs at a time.
These SMRT Cells are nanofabricated consumable substrates used in
conjunction with the DNA Sequencing Kit for automated processing on the
PacBio RS system.

PHOSPHO LINKED NUCLEOTIDE

For each of the nucleotide bases, there are four corresponding fluorescent dye
molecules that enable the detector to identify the base being incorporated by the
DNA polymerase as it performs the DNA synthesis. The fluorescent dye
molecule is attached to the phosphate chain of the nucleotide. When the
nucleotide is incorporated by the DNA polymerase, the fluorescent dye is
cleaved off with the phosphate chain as a part of a natural DNA
synthesis process during which a phospho diester bond is created to elongate the
DNA chain. The cleaved fluorescent dye molecule then diffuses out of the
detection volume so that the fluorescent signal is no longer detected.

PACBIO RS II
The instrument features high performance optics, automated liquid handling,
and an environmental control center, all directed through an intuitive
touchscreen interface. The computational brain responsible for primary data
analysis, called the Blade Center, is also included. This allows for seamless
integration of performance enhancements through chemistry and software
advances.
The instrument contains three primary user access points:

RS Touch uses a touchscreen interface to start and monitor runs at the

instrument.

Reagent and Sample Drawer for plate loading.

SMRT Cell and Tip Drawer for consumables loading.

Throughout the DNA sequencing process, the PacBio RS II uses SMRT
Technology to record light pulses emitted as a byproduct of nucleotide

incorporation. These signals are delivered in real time to the primary analysis
pipeline, housed entirely on the Blade Center

Run design and management can be done easily and efficiently through the RS
Remote and RS Touch user interfaces.
RS Touch
RS Touch, the integrated touchscreen interface, is your primary interaction
point with the PacBio RS II instrument. It guides you through instrument
loading and sequencing initiation. Intelligent features, including systemwide
barcode tracking, provide efficient management and high-confidence
performance. Throughout the single molecule, real-time (SMRT) sequencing
process, the RS Touch visibly monitors the overall system, including each
SMRT Cell, from preparation through quality assessment. The monitoring
function also displays a real-time sequencing video and expected time to run
completion.

RS Remote
The RS Remote is a Windows-based application that allows you to design and
monitor sequencing runs as well as view primary analysis results, directly from
your desktop. Here, you can define data analysis parameters, including content
and protocol settings, as well as set up automated secondary analysis for custom
data management convenience. The system also has build-in intelligence that
adjusts to provide the user constant feedback on the time and components
needed to complete a run.
Primary Analysis
Once sequencing is initiated, the systems computational Blade Center performs
real-time signal processing, base calling and quality assessment. Primary
analysis data, including readlength, distribution, polymerase speed and quality
measurement is streamed directly to the secondary analysis software. This data,
as well as trace and pulse data, are also available through the RS Touch and RS
Remote interfaces for quick assessment of a sequenced SMRT Cell.

DATA ANALYSIS
SMRT Portal
SMRT Portal is an open-source, browser-based application that allows to create,
submit and monitor secondary analysis jobs as well as view and download the
results. With this tool, you can align reads to a reference sequence or assemble
reads into a de novo genome.
SMRT View
SMRT View is a scalable and extensible application for graphical data
exploration and discovery that allows you to visualize and interact with
secondary analysis sequencing data. A flexible, application-oriented user
interface provides you with a broad range of ways to navigate and explore the
results of your experiments, supporting simple discovery of patterns in your
data. Interactive graphical representations of variants, quality values, and other
metrics, along with rich annotations allow you to rapidly validate your results
by visualizing genome-scale data sets down to the SNP level. SMRT View,
which includes out-of-the-box support for Sequencing and De Novo workflows,
is the first data visualization application that can visualize kinetics and structure
information unique to PacBio's SMRT technology.

Data analysis of pacbio RS uses various algorithms for different analytical

purposes such as
Hierarchical Genome Assembly Process (HGAP)
Quiver
BLASR (Basic Local Alignment with Successive Refinement)
AHA (A Hybrid Assembler)
Allora
Rare and Compound Variants

APPLICATIONS
Applications of Single Molecule Real Time Sequencing involves broad areas of
genomic research such as
De novo genome sequencing
Resequencing
Methylation detection
Full isoform sequencing
In Vitro Diagnostics.

REFERENCE
http://www.pacificbiosciences.com/
http://www.biomedcentral.com/1471-2164/13/341
https://www.youtube.com/playlist?list=PLFAB87D059EC592E9
SMRT Technology Overview
http://www.nature.com/nmeth/journal/v7/n6/abs/nmeth.1459.html