THE SEQUENCE OF SEQUENCERS. a journal presentationpptx

sequencing technique

1. THE SEQUENCE OF SEQUENCERS: THE HISTORY OF SEQUENCING DNA James M. Heather , Benjamin Chain (Genomics)

2. Introduction- “Knowledge of sequences could contribute much to our understanding of living matter” – Frederick Sanger This paper deals with how researchers throughout the years have addressed the problem of how to sequence DNA and characteristics that define each generation of methodologies for doing so.

3. First –generation DNA sequencing Watson and Crick solved 3D structure of DNA in 1953. It contributed to a conceptual framework for DNA replication and encoding proteins in nucleic acids Ability to read or sequence DNA was difficult as unlike proteins DNA molecules were longer and made of fewer units similar to one another I nitially pure RNA which were shorter and single stranded were used for sequencing . RNAse enzyme was already known and available by that time.

4. In 1965 Robert Holley’s lab was first to sequence whole nucleic acid sequence of alanine tRNA from S. cerevisiae . Fred Sanger parallelly developed related techniques based on detection of radiolabeled partial digestion fragments after 2D fractionation. Walter Fiers ’ lab produced first protein coding gene sequence of the coat protein of bacteriophage MS2 in 1972. Making use of observation that Enterobacteria phage lambda possessed 5’ overhanging ‘cohesive’ ends Ray Wu and Dale Kaiser used DNA polymerase to fill ends with radioactive nucleotides each type one at a time and measuring incorporation to deduce sequence.

5. Later primer was used to incorporate radioactive nucleotides to infer the order of nucleotides anywhere not just the end termini of phage. Next big change came when polyacrylamide gels replaced 2D fractionation(electrophoresis and chromatography) which gave better resolving power In mid 1970s Alan Coulson and Sanger came up with ‘plus and minus’ system- This had a ‘plus’ reaction where single type radiolabeled nucleotide present thus all extensions will end with that base. ‘minus’ reaction in which three are used which produced sequence upto position before next missing nucleotide. Running the products in polyacrylamide gel and comparing 8 lanes One gets the position of nucleotides.(exception homopolymer)

6. Sanger and colleagues sequenced 1 st DNA genome of bacteriophage X174. Maxam Gilbert had significantly different approach- using chemicals to break chains at specific bases. This technique was widely adopted and might be considered real birth of “first generation” DNA sequencing. But the major breakthrough came in 1977 with Sangers chain termination or dideoxy technique.

7. ddNTPs lack 3’ OH group therefore cannot form bond with 5’ phosphate of next dNTP. Radiolabelled ddNTPs randomly incorporated as strand extends halting further progression. 4 parallel reaction with 4 different ddNTPs and running on 4 lanes of polyacrylamide gel using autoradiograpy one could infer sequence in original template. Accuracy , robustness and ease of use led to Sanger sequencing to become the most common technology in DNA sequencing for years to come.

8. In following years radiolabeling was replaced by fluorometric detection. This allowed reaction to occur in one vessel instead of four and improved detection through capillary based electrophoresis. This techniques led to first generation DNA sequencing machines produce reads less than 1kb.

9. Development of PCR and RDT helped to generate high pure DNA required for sequencing. Klenow fragment DNA polymerase lacking 5’ to 3’ exonuclease activity was used to incorporate ddNTPs originally but better polymerase were genetically modified ABI PRISM dideoxy sequencer developed by Leroy Hood (Applied Biosystems) allowed simultaneous sequencing of hundreds of samples came to be used in Human Genome Project.

10. Second-generation DNA sequencing This method markedly differ from dideoxy method as it did not infer nucleotide identity using radio/fluorescently labelled dNTPs. Here scientists used luminescent method for measuring pyrophosphate synthesis which is two enzyme process- ATP sulfurylase is used pyrophosphate to ATP which is then used as substrate for luciferase, producing light proportional to amount of pyrophosphate. Both Sanger and pyrosequencing method are sequence by synthesis (SBS) techniques as both uses DNA polymerase.

11. Pyrosequencing could be performed using natural dNTPs without modification and observed in real time (no use of electrophoresis) Pyrosequencing was later licensed to 454 Life Sciences (purchased by Roche) which provided mass parallelization of sequencing greatly increasing the amount of DNA that can be sequenced in one run. Number of parallel sequencing techniques sprung up following success of 454, most important being Solexa method of sequencing (acquired by Illumina) Video demonstration

12. SOLiD system from Applied Biosystems – did not use polymerase thus sequenced not by synthesis method Though the above technology was cheaper in cost per base basis it was not able to produce read length and depth of Illumina machine. Ion Torrent (Life Technologies product) a post light sequencing technology as it uses neither fluorescence nor luminescence the nucleotide incorporation is measured not by pyrophosphate release but by pH difference caused by release of protons during polymerisation using metal oxide semiconductor technology.

13. Capabilities of DNA sequencers have grown at a rate even faster than computing revolution described by Moore’s law; the complexity of microchips doubles approximately every two years while sequencing capabilities between 2004 and 2010 doubled every five months. Illumina sequencing platform has been the most successful to the point near monopoly and thus can be considered to have made the greatest contribution to second generation DNA sequencers.

14. Third-generation DNA sequencing We consider third generation technologies to be those capable of sequencing single molecules, negating the requirement for DNA amplification shared by all previous technologies. Single molecule technology(SMS) developed by Stephen Quake( Helicoes BioSciences ) Which later developed as single molecule real time (SMRT) platform from Pacific Biosciences

15. Perhaps the most anticipated area of third generation DNA sequencing is the promise of nanopore sequencing. Oxford Nanopore Technologies was the first company offering nanopore sequencer. Video demonstration Ebola virus in Guinea was sequenced in two days after sample collection by Joshua Quick and Nicholas Loman using nanopore sequencers.

16. Conclusion- DNA sequencing has been compared to new microscope opening avenues to biological research at the most fundamental level it is how we measure one of the major properties by which terrestrial life forms can be defined and differentiated from each other. Researchers moved from lab to computer from pouring over gels to running code. Understanding of this history can provide appreciation of current methodologies and provide insights for future ones.

17. THANK YOU…….