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Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
Comparison of three High-throughput sequencing techniques
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Comparison of three High-throughput sequencing techniques

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  • 1. Comparison of three Highthroughput sequencing techniques Speaker P. RAMESH Ph.D
  • 2. HIGH-THROUGHPUT SEQUENCING 3 sequencing methods:  MegaBACETM Method 454 sequencingTM Single Molecule Real Time (SMRTTM) DNA sequencing
  • 3. MegaBACETM Method  First PCR amplification  After the amplification DYEnamic™ ET dye terminators and Thermo SequenaseTM II DNA Polymerase is used  Polymerase incorporates dNTPs in to the newly synthesized DNA strand during a PCR reaction  Dye terminators are fluorescently dye labelled ddNTPs that terminates further elongation of newly synthesized DNA strands
  • 4. Cont…. Each of the four ddNTPs has different dye labels Dye marked fragments are separated in the MegaBACE instrument, which is a fluorescence based detection capillary electrophoresis system that separate fragments according to their size
  • 5. 454 SEQUENCINGTM METHOD First developed in Sweden in the 90s (Ronaghi et al.,1998) An Emulsion based method, amplify DNA fragments invitro together with sequencing method (Pyrosequencing) Nucleic acids are randomly split into fragments 300-800 bp in length Speical adaptor (Biotinylated) are ligated to the fragments
  • 6. Cont…. Adaptors consist of two different primers, which are specific for 3’ and 5’ end of the fragments Individual fragments are ligated to special DNA capture beads (Streptavidin) against using the adaptors Beads are placed in heat stable water-in-oil emulsion & become captured in the water droplets Droplets contains PCR reagents After amplification the emulsion is broken from the beads
  • 7. Cont….  Beads which are carrying DNA fragments incubated with DNA bead Incubation Mix (DNA polymerase)  Loaded onto PicoTiter plate device for sequencing  PicoTiter plate is a fibre optic slide consists of open wells, that are so small, (75*10-12 l) that one bead can fit one well  Smaller enzymatic beads (Sulfurylase & Luciferase) are also loaded into the slide  PicoTiter plate is placed in the sequencing instrument (Sequence reagents, buffers & nucleotides)
  • 8. Single Molecule Real Time (SMRTTM) DNA sequencing Method Built on single sequencing by synthesis with fluorescent based detection  Sequence procedure takes place on SMRTTM chips  On each chip there is thousands of Zero-mode waveguides (ZMV)  ZMV are cylindrical holes just a few tens of nm in diameter  Holes perforate a thin metal film i.e supported by a transparent substrate
  • 9. Cont…. When laser light comes through the transparent substrate, the wavelength of the light is too large to pass through the small ZMVs Light does not stop directly at the opening of the ZMVs, instead attenuated light penetrates the lower 20-30nm of each ZMV A single DNA polymerase is attached to the transparent substrate at the bottom of each ZMV Phospholinked nucleotides are introduced into the ZMVs Each base carries a different coloured fluorophore
  • 10. Cont…. When a nucleotide is incorporated into the DNA strand, the polymerase holds the dye marked nucleotide in the ZMV’s detection volume for tens of milliseconds This creates a flash of bright light that can be detected light emitted by the fluorephores passes through a prism that deflects the light according to its colour. The light is thereafter transferred to a single-photon sensitive CCD array Position of the deflected light reveals which base that was creating the signal
  • 11. SYSTEM PERFORMANCES Throughput  MegaBACE simultaneosuly analyzes 384 DNA samples in one run, it can sequence 1920 templates within 8 hrs & generate 2 million bases  454 sequence has an average yield of 400 000 reads/run & generates more than 100 million bases/7.5 hrs run  SMRT can sequence 3000 templates/run, can improved 100 billion bases/hr
  • 12. Time Cont….  MegaBACE can run less than 2 hrs  454 sequence, the whole procedure takes 20 hrs & instruments run time is 7.5 hr  SMRT will take just a few minutes; it incorporates bases with a speed of 10 bases/sec DNA requirments  MegaBACE: 250-500 ng of plasmid or M13 DNA or 30-60ng of PCR prodcut is required  454 Sequence: 10-50 ng DNA & have conc.5ng/µl or more  SMRT: DNA requirement is not clear
  • 13. Read-length  MegaBACE: Avg read length is 500 bases. If using a slower 3 hr run time read lengths upto 1000 bases can be obtained  454 has an average read length of 200-300 bases. In 2008 it should be more than 400 bases (Roche Applied ScienceTM)  SMRT has read-length of 1500 bases & it can reach 10000 bases or more (Pacific BioscienceTM 2008)

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