1. Comparison of three Highthroughput sequencing techniquesSpeaker P. RAMESH Ph.D

2. HIGH-THROUGHPUT SEQUENCING3 sequencing methods: MegaBACETM Method454 sequencingTM Single Molecule Real Time (SMRTTM) DNA sequencing 3. MegaBACETM Method First PCR amplification After the amplification DYEnamic ET dye terminators andThermo SequenaseTM II DNA Polymerase is used Polymerase incorporates dNTPs in to the newly synthesizedDNA strand during a PCR reaction Dye terminators are fluorescently dye labelled ddNTPs thatterminates further elongation of newly synthesized DNAstrands 4. Cont. Each of the four ddNTPs has different dye labelsDye 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 lengthSpeical 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 fragmentsIndividual fragments are ligated to special DNA capture beads (Streptavidin) against using the adaptorsBeads are placed in heat stable water-in-oil emulsion & become captured in the water dropletsDroplets 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 ZMVsLight does not stop directly at the opening of the ZMVs, instead attenuated light penetrates the lower 20-30nm of each ZMVA single DNA polymerase is attached to the transparent substrate at the bottom of each ZMVPhospholinked 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 ZMVs detection volume for tens of millisecondsThis 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 arrayPosition 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. TimeCont. 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/secDNA 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/l 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)