Next generation sequencing

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Next generation Sequencing

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  • Substrate attachment; dibase probes Make sequencing library by shearing and adapter ligation Attach DNA fragments to beads and amplify polonies in emulsion Attach beads to slide
  • Next generation sequencing

    1. 1. NEXT GENERATION SEQUENCING METHODS Course teacher By Dr. N. Senthil Nidhi Singh (09-607-010)
    2. 2. WHAT IS SEQUENCING?  “Sequencing” means finding the order of nucleotides on a piece of DNA .  Nucleotide order determines amino acid order, and by extension, protein structure and function.  An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal.
    3. 3.  The sequencing of the reference human genome was the capstone for many years of hard work spent developing high- throughput.  Scenario is rapidly changing owing to the invention and commercial introduction several revolutionary approaches to DNA sequencing, the so-called next-generation sequencing technologies  Major impact on our ability to explore and answer genome- wide biological questions.  Not only changing our genome sequencing approaches and the associated timelines and costs, but also accelerating and altering a wide variety of types of biological inquiry that have historically used a sequencing-based readout,
    4. 4. SANGER DDNTP CHAIN TERMINATION SEQUENCING Daniel, 2009
    5. 5. MAXAM-GILBERT METHOD
    6. 6. COUNTRIES CONTRIBUTION TO SEQUENCING 2010
    7. 7. MAJOR SEQUENCING CENTERS
    8. 8. ADVANCEMENT IN SEQUENCING 2010
    9. 9. NEXT GENERATION SEQUENCING TECHNIQUES  454 Sequencing (Pyro sequencing)  ABI Solid (Sequencing By Ligation)  Helicos (True Single Molecule Sequencing)  Nanopore (Single Base Resolution)  Illumina /Solexa: Genetic Analyzer
    10. 10. ROCHE (454) GS FLX  In 2005, Developed by 454 Life Sciences.  Follows Sequencing-By-Synthesis chemistry.  Initially with 96 sample capacity in parallel in a microtiter plate.  Now Picotiter plates & Streptavidin beads are used allowing about 1 million seq reads.  GS FLX Titanium series sequence 400-600 million bases per 10-hour run. Wilhelm , 2009
    11. 11. •454 is based pyrosequencing on the generation of light signal through release of pyrophosphate (PPi) on nucleotide addition. DNAn + dNTP  DNAn+1 + PPI •PPi is used to generate ATP from adenosine phosphosulfate (APS). APS + PPI  ATP •ATP and luciferase generate light by conversion of luciferin to oxyluciferin. Principle Wilhelm ,2009
    12. 12. SEQ-BY-SYNTH 454 LIFE SCIENCES PLATFORM Wilhelm , 2009
    13. 13. ABI SOLID  Sequencing by Oligo Ligation and Detection.  Also called as ‘2 base encoding’.  Developed by Applied Biosystems in Autumn 2007.  In 2008,updated version ABI SOLid 2.0 platform is commercialized.  Polystyrene beads are used. Shendure et al, 2008
    14. 14. SEQUENCING BY LIGATION DNA fragment ligated to 1 µm Polystyrene/magnetic bead. Amplification by Emulsion PCR Universal sequencing primer complementary to adaptor Shendure et al, 2008
    15. 15. SOLiD: Sequencing ligation cycles Shendure et al, 2008
    16. 16. Decoding of paired bases is done by determing the overlapping bases from each pair. Sequences are Computated and sequence result is displayed Shendure et al, 2008
    17. 17. HELISCOPE (TRUE SINGLE MOLECULE SEQUENCING)  Developed by Helicos Biosciences in 2007.  No Amplification needed  Single molecule detection  Homopolymer stretches(solved)  Expensive Detection Wilhelm , 2009
    18. 18. Wilhelm, 2009
    19. 19. ILLUMINA GENOME ANALYZER •single molecule amplification •starts with a Illumina-specific adapter library •performed by an automated device called Cluster Station •uses DNA polymerase to produce multiple DNAcopies, •utilizes a sequencing by synthesis approach •the nucleotides carry a base-unique fluorescent label •the 3-OH group is chemicall blocked such that each incorporation isa unique event Shendure et al, 2008
    20. 20. •Imaging step is followed with each base incorporation •each flow cell lane is imaged in three 100-tile segmentby the instrument optics at a cluster densitper tile of 30,000 • After each imaging step the 3-OH blocking group is chemically remove • quality checking pipeline evaluates the Illumina data from each run • A base-calling algorithm assigns sequences and associated quality values to each read
    21. 21. Shendure et al, 2008
    22. 22. NANO-PORE DNA SEQUENCING  Nanopore is a nanoscale pore in an electrically insulating membrane - a pore in a solid-state membrane (most commonly Si3N4)  Nanofabricated pores that are contained in biological or inorganic membranes might allow for the sequencing of individual molecules of DNA. Eugene, 2005
    23. 23.  As the molecule translocates across the membrane , the identity of individual nucleotides in the molecule of DNA is determined using ionic current or transverse current . Eugene, 2005
    24. 24.  DNA translocates across the nanopore and produces a change in the transverse current between two electrodes that are on either side of the nanopore.  Nanopores can also operate using changes in ionic current, that is, the flow of positive ions in the opposite direction to the negatively charged DNA translocating the nanopore. Eugene, 2005
    25. 25. Sequencing Chemistry Read Length Cost per instrument Total output TIME/run Accuracy Sanger Sequencing- By-Synthesis 800 bp -- 700 Mb-1 Gb -- 99 % 454 GS Sequencing - by-Synthesis 300-800 bp $500,000 4 Gb 7 hr 99 % ABI SoLiD Sequencing- by-Ligation 25-35 bp $591,000 3-10 Gb 4.5 Days 99.94 % Heliscope True Single Molecule Sequencing 30-55 bp $1,350,000 21-35 Gb 14 Days 99.99 % Nanopore Single Molecule Resolution 5.4 kb -- 35-76 Gb 11 hr 99.9 % COMPARISION OF SEQUENCING METHODS William Stafford Noble , 2010
    26. 26. CONCLUSION •The new method are fast as it takes about 1 day to sequence 100 million bases. • Inexpensive due to lost per base sequencing. • Accurate by using a sequencing array and high accuracy can be achieved by analyzing several time series.
    27. 27. APPLICATION Avak et al., 2008
    28. 28. COMPANIES DETAIL Elim Biopharmaceuticals, Inc leading , in the San Francisco Bay Area Elim has pioneered many new features in DNA sequencing services Bioaxis DNA Research Centre private limited , top level CRO in life sciences , vast range of services in Biotechnology, Bioinformatics and Clinical Research NanoBioServices Chandigarh, India NanoBioServices is a contract research and contract manufacturing organization Xcelris Gujarat, India Xcelris provides bio-analytical services., includes SNP analysis, m-RNA profiling Genewiz, Inc. South Plainfield, New Jersey Genewiz, Inc. is a contract research organization specializing in DNA sequencing, molecular biology DNA Analysis, LLC Cincinnati, Ohio DNA Analysis, LLC provides complete sequencing and fragment analysis service including analysis MWG Biotech Pvt. Ltd Karnataka, India MWG Biotech Pvt. Ltd. Focuses on genotyping and bioinformatics.

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