This document discusses DNA sequencing methods, both current and developing technologies. It begins by explaining Sanger sequencing and how fluorescent dyes and thermal cycling improved it. High-throughput short and long-read sequencing methods are then outlined, including Illumina, Ion Torrent, Nanopore, and SMRT sequencing. Developing methods like tunneling currents, hybridization, and microscopy techniques are also mentioned. Overall, the document provides a comprehensive overview of the major DNA sequencing techniques used today and those under investigation.

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Presented by: Sepideh Saroughi

2. DNA sequencing is the process of determining
the nucleic acid sequence – the order of
nucleotides in DNA
Medical diagnosis
Biotechnology
Forensic biology
Virology
1970s : two-dimensional chromatography,
fluorescence-based sequencing methods
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3. Sequencing of full genomes
Basic methods
Large-scale sequencing and de novo sequencing
High-throughput methods
Long-read sequencing methods
Short-read sequencing methods
Methods in development
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4. Sequencing of full genomes
Whole genome sequencing
Whole genome sequencing (WGS), also known as full
genome sequencing
Saliva, Epithelial cells, Bone marrow, Hair, Seeds,
Plant leaves
High-throughput sequencing (HTS) methods
Personalized Medicine
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5. Basic methods
1. Maxam-Gilbert sequencing
This method is based on nucleobase-
specific partial chemical modification of
DNA and subsequent cleavage of the DNA
backbone at sites adjacent to the modified
nucleotides
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6. 2. Chain-termination methods
Single-stranded DNA molecules
Polyacrylamide gel
Short oligonucleotide + template = primer
Dideoxynucleotides (ddNTPs: ddATP, ddCTP, ddGTP,
and dNTP)
Not all DNA polymerases can be used for sequencing
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8. The most significant innovations in Sanger
sequencing have been:
1. The development of fluorescent dyes
2. The use of thermal-cycle sequencing
3. Software developments to interpret and analyze
the sequences
Licor, Amersham, MilliGen, Perkin Elmer and
Dupont, all of them except the AB machines
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9. Large-scale sequencing and denovo
sequencing
Very long DNA pieces
Shotgun sequencing
Analysis of DNA sequences longer
than 1000 base pairs
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10. High-throughput methods
 Long-read sequencing methods
1. Single molecule real time (SMRT) sequencing
2. Nanopore DNA sequencing
 Short-read sequencing methods
1. Massively parallel signature sequencing (MPSS)
2. Polony sequencing
3. 454 pyrosequencing
4. Illumina (Solexa) sequencing
5. SOLiD sequencing
6. Ion Torrent semiconductor sequencing
7. DNA nanoball sequencing
8. Helicos single molecule fluorescent sequencing
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11. Long-read sequencing methods
1. Single molecule real time (SMRT) sequencing
Real-time sequencing also enables read lengths to be
longer
Read lengths of up to 20 000 bp
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12. 2. Nanopore DNA sequencing
Using nanopore sequencing, a single molecule of DNA or RNA
can be sequenced without the need for PCR amplification or
chemical labeling of the sample
MinION Mkl
Problem:
1. reduce the high error rates of
base
2. optimize the speed of DNA
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13. Short-read sequencing methods
1. Massively parallel signature sequencing (MPSS)
MPSS is a method for
determining expression levels
of mRNA by counting the
number of individual mRNA
molecules produced by each
gene.
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14. 2. Polony sequencing
Multiplex sequencing technique
Used to “read” millions of immobilized DNA sequences in
parallel
Paired-end tags library
DNA template is of 135 bp in length with two 17–18 bp
paired genomic tags separated
The current read length of this technique is 26 bases per
amplicon and 13 bases per tag, leaving a gap of 4–5 bases in
each tag.
The protocol of Polony sequencing can be broken into three
main parts, which are the paired end-tag library
construction, template amplification and DNA sequencing.
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15. Polony sequencing
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16. 3. 454 pyrosequencing
High throughput , Second generation
A single strand of DNA with a length of 400-500bp
The 4 required enzymes are: DNA Polymerase, ATP
Sulfurylase, Luciferase, Apyrase
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17. 4. Illumina (Solexa) sequencing
Reversible
Identification of single nucleotides
Illumina sequencing technology works in three basic
steps: amplify, sequence, and analyze.
Purified DNA
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18. 5. SOLiD sequencing
Next-generation DNA sequencing
99.94%
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19. 6. Ion Torrent semiconductor sequencing
Hydrogen ions
Sequencing by synthesis
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20. A major advantage of the system is that no camera, light
source or scanner is needed
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21. 7. DNA nanoball sequencing
High throughput sequencing
Genomic sequence
Rolling circle replication
Fluorescence
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22. 8. Helicos single molecule fluorescent sequencing
 The first commercial NGS
 Single molecule fluorescent sequencing
 M13 bacteriophage
 Successfully sequenced the human genome
 Clinical evaluation & Sequenced RNA
 2 STEP
1. Preparing the DNA
(Fragmenting the DNA; Tailing; Blocking)
2. DNA sequencing
(Sample loading; Filling and locking;
Sequencing)
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23. Methods in development
1. Tunnelling currents DNA sequencing
2. Sequencing by hybridization
3. Microfluidic Sanger sequencing
4. Microscopy-based techniques
5. RNAP sequencing
6. In vitro virus high-throughput sequencing
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24. 1. Tunnelling currents DNA sequencing
Tunneling current measurements in liquid have been considered
a promising way to identify the sequence of base molecules in
RNA and DNA
A pair of metal electrodes
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25. 2. Sequencing by hybridization
 Small changes relative to a known DNA sequence
 DNA chips , microarrays , synthetic oligonucleotides
 Affymetrix
 NABsys
3. Microfluidic Sanger sequencing
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26. 4. Microscopy-based techniques
Microscopic evaluation of specimens
Symptomatic patient
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27. 5. RNAP sequencing
RNA polymerase (RNAP)
Polystyrene bead
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28. 6. In vitro virus high-throughput sequencing
Protein interactions
Combination of 454 pyrosequencing and an in vitro virus
mRNA display method
7. Sequencing with mass spectrometry
MALDI-TOF MS
Size
Easily detect differences between RNA fragments
Single-nucleotide polymorphisms in human
Up to 100 base pairs
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29. Next-generation sequencing
Library (Breakage of DNA, Immobilization, Amplification)
Up of thousands or millions of DNA fragments
Single experiment
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31. Methods
There is no electrophoresis or any other fragment separation
step.
1. Reversible terminator sequencing
2. Pyrosequencing
Illumina sequencing 31

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