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.
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.
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
17. Automation of Sanger sequencing
SeqStudio Genetic Analyzer
4 capillaries
3500 Series Genetic Analyzer
8–24 capillaries
3700 Series Genetic Analyzer
48–96 capillaries
18.
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
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
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
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39. CREDITS: This presentation template was created by Slidesgo,
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THANKS
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