Next generation sequencing

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NEXT GENERATION SEQUENCING – A MOLECULAR MICROSCOPE (A TOOL TO GET A FIRST LOOK) Presented By UZMA JABEEN MSc. Microbiology

Transcript of Next generation sequencing

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NEXT GENERATION SEQUENCING – A MOLECULAR MICROSCOPE

(A TOOL TO GET A FIRST LOOK)

Presented ByUZMA JABEEN

MSc. Microbiology

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CONTENT INTRODUCTION HISTORY AND FUTURE OF SEQUENCING SANGER SEQUENCING NEXT GENERATION SEQUENCING WORKFLOW SEQUENCING APPROACH SEQUENCING PLATFORMS NEXT NEXT GENERATION SEQUENCING NGS APPLICATIONS CONCLUSION FUTURE TASK

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DISCOVERY OF DNA STRUCTURE

We have discovered the secret of life”February 28, 1953

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INTRODUCTION DNA was demonstrated as the genetic material by Oswald Theoder Avery in 1944.

Its double helical strand structure composed of four bases was determined by James D. Watson and Francis Crick in 1953 leading to Central Dogma Of Molecular Biology

DNA Has Only Two JobsI. It serves as a store of information ,ensuring that information is passed on to

each new cell upon division (and the next generation)II. It directs the synthesis of proteins, which are necessary to carry out the

functions of a living organism

We have 3 billion bases arrayed in a unique order, with ~20,000 genes that direct the synthesis of all the proteins that comprise us

continued………

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“Sequencing” DNA is simply the elucidation of the order of the bases in an organism’s DNA strand.

DNA sequencing technologies could help biologist and health care providers in a broad range of applications such as molecular cloning , breeding, finding pathogenic genes, comparative and evolution studies.

These technologies should be fast accurate, easy to operate and cheap.

In past thirthy years, DNA sequencing technologies and applications have undergone tremendous development and act as the engine of the genome era.

It is necessary to look back on the history of sequencing technologies development to review the Next Generation Sequencing system to compare their advantages and disadvantages and to discuss various applications and also to evaluate the recently introduced Third Generation Sequencing technologies and applications.

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1st GENERATION SEQUENCINGSANGER SEQUENCING

In 1977, Fredrick Sanger developed DNA sequencing technology which was based on chain termination method. It is also known as Sanger sequencing or dideoxy nucleotide chain termination sequencing.Because of its high efficiency and low radioactivity, Sanger sequencing was adopted as the primary technology in the first generation of laboratory and commercial sequencing application.

DIDEOXY NUCLEOTIDEDEOXY NUCLEOTIDE

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Sanger sequencing method

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NEXT GENERATION SEQUENCING

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Next Generation Sequencing (NGS) or Massive Parallel Sequencing is any of the several high throughput approaches to DNA Sequencing using the concept of Massive Parallel Processing. Some of these technologies emerged in 1944-1998 and have been commercially available in 2005.These technologies uses miniaturized and parallelized platforms for sequencing one million to 43 billion short reads per instrument run.

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workflowPrepare a library

Amplification of library fragments

Run the amplified sequences on selected NGS Platform

Run type

Analysis of Data

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AMPLIFICATION OF LIBRARY FRAGMENTS

All NGS technologies replicates DNA before sequencing is done. No matter the method of sequencing, without a proper amount of amplification it is impossible to detect each base call.Amplification of library fragments can be done by two methods:

By Emulsion PCR By Bridge Amplification

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EMULSION PCREmulsion PCR is a PCR variation that some NGS technologies use to replicate DNA sequences. It is conducted on a beads surface within tiny water bubble floating on oil solution.

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BRIDGE AMPLIFICATIONAn amplification reaction that occurs on the surface of an illumina flow cell. During flow cell manufacturing, the surface is coated with a lawn of two distinct oligonucleotides referred to as “p5” and “p7”.In the first step of bridge amplification a single stranded sequencing library (with complimentary adapter ends) is loaded into the flow cell. Individual molecules in library bind to complimentary oligos as they flows across the oligo lawn. Priming occurs as the opposite ends of the ligated fragments bends over and bridges to another complimentary oligos on the surface. Repeated denaturation and extension cycle results in localised amplification of single molecules into millions of unique clonal clusters across the flow cell. This process also known as clustering occurs in an automated flow cell instruments

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PYROSEQUENCING

SEQUENCING BY REVERSIBLE TERMINATOR CHEMISTRY (SEQUENCING BY SYNTHESIS)

SEQUENCING BY LIGATION MEDIATED BY LIGASE ENZYME

PHOSPHOLINKED FLUROSCENT NUCLEOTIDE OR REAL TIME SEQUENCING

SEQUENCING APPROACHES

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PYROSEQUENCINGPyrosequencing is based on the detection of light signal upon incorporation of nucleotide by polymerase. Incorporation of dNTP releases pyrophosphate(PPi). In the presence of ATP sulfurylase and adenosine 5’-phosphosulfate (APS), PPi is converted to ATP. Luciferase-ATP mediates conversion of luciferin to oxyluciferin, which generates a light signal. Amount of light is proportional to the numberof ATP molecules, which is proportional to the number of released pyrophosphates, or, in

other words, incorporated nucleotides.

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SEQUENCING BY SYNTHESISSequencing By Synthesis (SBS) technology uses four fluorescently labelled nucleotide to sequences the tens of millions of clusters on flow cell surface in parallel . During each sequencing cycle, a single labelled dNTPs is added to the nucleic acid chain. The nucleotide label serve as “reversible terminator” for polymerization. After dNTPs incorporation, the fluorescent dye is identified through laser excitation and imaging then enzymatically cleaved to allow the next round of incorporation. As all four reversible terminator bound dNTPs (A,C,T,G) are present natural competition minimizes incorporation bias. Base calls are made directly from signal intensity measurement during each cycle, which greatly reduces raw error rate compare to other technologies. This result in highly accurate base by base sequencing that eliminate sequencing context specific errors enabling robust base calling across the genome, including repetitive sequence regions and within homopolymer region.

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SEQUENCING BY LIGATIONSBL is a straightforward enzymatic method of sequencing DNA. SBL uses known, universal sequences that flank an unknown genomic tag as anchor primer sites. An anchor primer is hybridized to one of these known regions, and a ligatable end (3' or 5' depending on the direction of desired sequencing) is available. An oligo, called a query primer, is then ligated to the end of the anchor primer. The query primer is a mix of oligos that are degenerate for all positions except a single position that is being sequenced, which allows the sequencing of a single position based on the design of the query primer. After sequencing a single position, the query primer and anchor primer are stripped from the DNA template, effectively resetting the sequencing. The process begins again, sequencing a different position by using a different query primer, and repeating until the entire sequence of the tag has been determined.

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ION SEMICONDUCTOR SEQUENCING

Similar to Pyrosequencing, but measures the release of H+ ions instead of pyrophosphates. More cost effective and time efficient.

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454 SEQUENCING/ ROCHE

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ILLUMINA SEQUENCING PLATFORMS

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APPLIED BIOSYSTEM SOLID SEQUENCING PLATFORM

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ION TORRENT SEQUENCING PLATFORM

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3rd GENERATION SEQUENCING OR NEXT

NEXT GENERATION SEQUENCING

(single molecule sequencing without PCR amplification)

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HELICOS TRUE SINGLE MOLECULE SEQUENCING

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SMRT DNA SEQUENCINGSINGLE MOLECULE REAL TIME SEQUENCING

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PACIFIC BIOSCIENCES SEQUENCING PLATFORM

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OXFORD NANOPORE SEQUENCING

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OXFORD NANOPORE SEQUENCING PLATFORM

MINION SYSTEM

GRIDION SYSTEM

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NGS APPLICATIONS

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CONCLUSION Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Gradually, sequencing is starting to become the standard technology to apply, certainly at the first step where the main question is “what's all involved”, “what's the basis”. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research, metagenomics agricultural and environmental science. Algorithms are continuously under development and it is thought that the future of this field lies with improving the statistics models used in algorithms as well as increasing the sensitivity of this algorithms in evaluating the probabilities of certain base combination. Many regulatory challenges and technical challenges remains but Next Generation Sequencing technology have evolved to where we can now implement them clinically.

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FUTURE TASK Standardization of NGS data and creation and maintenance of reference database. The applicability of NGS to clinical diagnostics and reference laboratories. Cost of technology Efficiency of correct identification and development of standard operating procedure.

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REFERENCES1. Next-Generation Sequencing Platforms ,Elaine R. Mardis Annu. Rev. Anal. Chem. 2013.

6:287–3032. THE HISTORY OF DNA SEQUENCING Miodrag J Med Biochem 32: 301–312, 2013

Review article3. Next generation sequencing technology: Advances and applications H.P.J. Buermans 1, J.T.

den Dunnen Biochimica et Biophysica Acta 20134. Next generation sequencing by Andy Vierstraete Ghent University june 20125. A window into third-generation sequencing Eric E. Schadt, Steve Turner and Andrew

Kasarskis Hum Mol Genet.2010 Oct 15;19(R2):R227-40. Epub 2010 Sep 21.6. An Introduction to Next Generation Sequencing by Illumina7. A review of DNA sequencing techniques Lilian T. C. Franc:a1, Emanuel Carrilho2 and

Tarso B. L. Kist3*Quarterly Reviews of Biophysics 35, 2 (2002), pp. 169–200. " 2002 Cambridge University Press

8. DNA Sequencing – Methods and Applications Edited by Anjana Munshi Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia

9. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific10. Biosciences and Illumina MiSeq sequencers Michael A Quail*, Miriam Smith, Paul

Coupland, Thomas D Otto, Simon R Harris, Thomas R Connor, Anna Bertoni, Harold P Swerdlow and Yong Gu BMC Genomics 2012, 13:341

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THANKS