Generations of sequencing technologies.

This presentation describe briefly some of the sequencing technologies in each of the first, second and third generation of sequencing.

1. ShadenAlharbi
Medical Laboratory Intern at Ministry of
National Guard Health Affairs
.
Generations of Sequencing
Technologies

2. OBJECTIVES
INTRODUCTION TO SEQUENCING01
02FIRST GENERATION SEQUENCING
03SECOND GENERATION SEQUENCING
04THIRD GENERATION SEQUENCING

3. Generations of Sequencing
Technologies

4. INTRODUCTION TO SEQUENCING 01

5. WHAT IS SEQUENCING?
•DNA sequencing process utilizes biochemical methods in order
to determine the correct order of nucleotide bases in a DNA
macromolecule using sequencing machines.
•Sequencing enabledthe completion of notable projects, such
as:
§The Human Genome Project
§The 1000 Genomes Project.

6. FIRST GENERATION SEQUENCING (FGS)
§Sanger chain termination
sequencing
§Maxam-Gilbert Chemical
sequencing
02

7. Fredrick Sanger
(1977, University of Cambridge)
Nobel Prize in Chemistry in 1980
The sole method for DNA sequencing for 3 decades
FIRST GENERATION SEQUENCING
Sanger Chain Termination SequencingMaxam-Gilbert Chemical Sequencing
AllanMaxam and
Walter Gilbert
(1976–1977, Harvard University)
•lesser technical
complexity
•lesser amount of
toxic chemicals used
•first automated
sequencing
technique

8. •Also know as: Chain termination sequencing or dideoxy method .
•Sequencing by synthesis method
•Reaction component:
§DNA template
§DNA primers
§DNA polymerase
§Four normal DNA nucleotides
§Four fluorescently labeled modified nucleotides
(ddATP, ddCTP, ddGTPand ddTTP).
SANGER SEQUENCING METHOD
8

9. Dideoxynucleotide
3’ OH group required for the extension is missing

10. Fluorescently Labeled Dideoxynucleotide

13. CHAIN TERMINATION
Chain terminates
at ddGTP
13

15. Shorter fragments
Longer fragments

16. Electropherogram

17. Automation of Sanger sequencing
SeqStudioGenetic Analyzer
4 capillaries
3500 Series Genetic Analyzer
8–24 capillaries
3700 Series Genetic Analyzer
48–96 capillaries

19. SECOND GENERATION SEQUENCING (SGS)
§454 PYROSEQUENCING
§ILLUMINA’S SEQUENCING
§SOLiDsequencer
§Ion Torrent Personal Genome
Machine (PGM)
03

20. •Three major SGS methods include:
•The Roche, 454 pyrosequencing system.
•The Illumina/SolexaGenome Analyzer.
•The Applied Biosystems, SOLiDTMSystem.
●SGS workflows involve:
1.Obtaining the nucleic acid of interest
2.Preparing a sequencing library, which involve enrichment of target sequences
3.Carrying out the sequencing on the chosen platform.
SECOND GENERATION SEQUENCING (SGS)
20

21. §Sequencing by synthesis method
§Amplification is carried by bridge PCR.
§Based on reversible dye terminators
§Reaction component:
§DNA template
§Adapters
§DNA polymerase
§dNTP
§Flow cell
§DNA primer
§Four fluorescently labeled 3’blocked reversible terminators
ILLUMINA SEQUENCING
21

22. 22
ILLUMINA SEQUENCING WORKFLOW

23. The flow cell
23

24. 24
ILLUMINA SEQUENCING WORKFLOW

25. 25
ILLUMINA SEQUENCING WORKFLOW
Incorporation
ImagingDeprotection
Sequencing
cycle

26. 3’ blocked reversible terminator
26Terminating functional groups
Site of fluorophore
cleavage
Residual linker structures

27. 27
ILLUMINA SEQUENCING WORKFLOW

28. Illumina (Solexa) Genome Analyzer
MiSeq
HiSeq
MiniSeq NextSeq
NovaSeq

29. 29

30. THIRD GENERATION SEQUENCING (TGS)
§Nanopore sequencing
§Pacific single molecule real time
(SMRT) DNA sequencing
04

31. §There are three important improvements in TGS platforms:
1.Increase in read length from tens of bases to tens of thousands of bases per
read.
2.Reduction of sequencing timefrom days to hours (or to minutes for real-time
applications).
3.Reduction or elimination of sequencing biasesintroduced by PCR amplification.
●The two most promising TGS technologies are:
§Nanopore DNA sequencing
§Oxford Nanopore
§Pacific single molecule real time (SMRT) DNA sequencing.
THIRD GENERATION SEQUENCING
31

32. ●Released the MinIONdevice in 2014 which is small size device.
●Low equipment cost.
●Sequencing of individual DNA molecules with long read lengths (up to 50,000 bp).
●Read accuracy ranging from 65%-88%.
●No sequencing-by-synthesis.
Oxford Nanopore sequencing (ONT)
32

33. The MinION device
●The smallest sequencing
device.
●Weight = 90 g.
●Flow cell with 512
channels.
●Default run time: 48-h
Flow cell
Nanopore

34. Nanoporesequencing
34

35. Pore Ionic current trace
35

36. Biology for anyone, anywhere
36

37. Sequencing in space
37

38. REFERENCES
●MetzkerML. Sequencing technologies —the next generation. Nat Rev Genet.2009;11(1):31–46.
http://dx.doi.org/10.1038/nrg2626
●Lu H, Giordano F, Ning Z. Oxford Nanopore MinIONSequencing and Genome Assembly. Genomics Proteomics
Bioinformatics. 2016;14(5):265–79.
http://dx.doi.org/10.1016/j.gpb.2016.05.004
●Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics.2016;107(1):1–8.
http://dx.doi.org/10.1016/j.ygeno.2015.11.003
●KchoukM, GibratJ, ElloumiM. Generations of Sequencing Technologies: From First to Next Generation. Biol
Med. 2017;9(3).
●Kulkarni S, Pfeifer J. Emerging DNA Sequencing Technologies. Clinical Genomics. Elsevier Inc.; 2015. 69–76 p.
http://dx.doi.org/10.1016/B978-0-12-404748-8.00005-8
●Masoudi-nejadA, NarimaniZ, HosseinkhanN. Next Generation Sequencing and Sequence Assembly
Methodologies and Algorithms. Springer; 2013.
●Hagemann IS. Overview of Technical Aspects and Chemistries of Next-Generation Sequencing. Clinical Genomics.
Elsevier Inc.; 2015. 1–20 p.
http://dx.doi.org/10.1016/B978-0-12-404748-8.00001-0
●Biorender.com 38

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