This document discusses the history and evolution of DNA sequencing technologies. It describes 3 generations of sequencing: 1) First generation sequencing involved Sanger chain termination sequencing; 2) Second generation sequencing included 454 pyrosequencing, Illumina sequencing, and SOLiD sequencing, which allowed massively parallel sequencing; 3) Third generation sequencing features long read lengths up to 50,000 bp from technologies like nanopore sequencing from Oxford Nanopore and single molecule real-time sequencing. The document provides details on the workflow and chemistry of various sequencing platforms.

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

2. OBJECTIVES
INTRODUCTION TO SEQUENCING 01
02
FIRST GENERATION SEQUENCING
03
SECOND GENERATION SEQUENCING
04
THIRD 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 enabled the 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 Sequencing
Maxam-Gilbert Chemical Sequencing
Allan Maxam 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, ddGTP and 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
SeqStudio Genetic 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
§ SOLiD sequencer
§ Ion Torrent Personal Genome
Machine (PGM)
03

20. • Three major SGS methods include:
• The Roche, 454 pyrosequencing system.
• The Illumina/Solexa Genome Analyzer.
• The Applied Biosystems, SOLiDTM System.
● 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
Imaging
Deprotection
Sequencing
cycle

26. 3’ blocked reversible terminator
26
Terminating 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 time from days to hours (or to minutes for real-time
applications).
3. Reduction or elimination of sequencing biases introduced 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 MinION device 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. Nanopore sequencing
34

35. Pore Ionic current trace
35

36. Biology for anyone, anywhere
36

37. Sequencing in space
37

38. REFERENCES
● Metzker ML. 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 MinION Sequencing 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
● Kchouk M, Gibrat J, Elloumi M. 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-nejad A, Narimani Z, Hosseinkhan N. 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

39. CREDITS: This presentation template was created by Slidesgo,
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