next generation seqencing

1. Present by : Yaser mohassel

2. History
 1869 – Discovery of DNA
 1977 – Sanger sequencing invented
 1986 – First automated sequencing machine
 1990 – Human Genome Project started
 2005 – First “next-generation” sequencing instrument
 2013– >10,000 genome sequences in NCBI database

3. Sequencing methods
Two basic methods for DNA sequencing:
A- Chemical cleavage method (Maxam and Gilbert, 1977)
- Four different chemicals, one for each base
- A set of DNA fragments of different sizes
-DNA fragments contain up to 500 nucleotides
B- Enzymatic method (Sanger)

4. Sanger sequencing: chain termination method
• Uses DNA polymerase
• All four nucleotides, plus one dideoxynucleotide (ddNTP)
• Random termination at specific bases
• Separate by gel electrophoresis

5. Sanger sequencing: chain termination method
Incorporation of di-deoxynucleotides terminates DNA elongation
Individual reactions for each base

6. Sanger sequencing: chain termination method

7. Sanger sequencing: chain termination method

8. Separation of fragments by gel electrophoresis

9. Sanger sequencing: dye terminator sequencing

10. MAXAM & GILBERT METHOD
 Maxam and Gilbert-1977
 Chemical Sequencing
 Treatment of DNA with certain Chemicals DNA cuts into
Fragments Monitoring of sequences

11. 1. The double-stranded fragment to be sequenced is isolated and
radioactively labeled at the 5’-ends with 32P.
2. The fragment is then cut with restriction enzyme and thus the
label is removed from one end.
3. The fragment of DNA with one end labeled is denatured.
4. Four identical samples of these end-labeled DNA restriction
fragments are subjected to chemical cleavage at different
chemical nucleotides.
5.There are four specific sets of chemical reactions that selectively
cut the DNA backbone at G, A+G, C+T, or C residues.
– G only: Dimethyl sulphate(DMS)
and piperidine
– A+G :Formic acid, piperidine
– C+T : Hydrazine, piperidine
– C only : Hydrazine,NACL,
piperidine
Maxam-Gilbert method

12. Chemical degradation method (Maxam–Gilbert method)

13. The Human Genome Project
 The biggest challenge for the life sciences
Some reasons for studying Human genome:
– Better medical practice
– High-quality diagnosis of diseases
– Understanding of evolution fully
– Improvement in biological research and forensic science
– Improvement in agriculture etc.

14. Next generation sequencing (NGS)
• It started in 2005 by Roche seqencer
And after in 2006 Illumina(an American company) entered the field by
purchasing SOLEXA
NGS platforms generate millions of reads and billions of base calls each run
• There are four main sequencing methods
– Pyrosequencing (454)
– Reversible terminator sequencing (Illumina)
– Sequencing by ligation (SOLiD)
– Semiconductor sequencing (Ion Torrent)
• NGS reads are typically short (<400 bp)
• Next generation sequencing is used for a range application including
– sequencing whole genomes
– sequencing specific genes or genomic reagions
– gene expression analysis
– study of epigenetics

15. Low use methods
•MPSS
•Helioscope
•(RNA Polymerase) RNAP
•Nanoball
•Nanopore

16. Next-gen sequencing technologies

17. in 2005

18. Next-gen sequencing:
shotgun library preparation

19. (Emulsion PCR (emPCR
 Emulsion PCR is a method of clonal amplification which allows
for millions of unique PCRs to be performed at once through
the generation of micro-reactors.

20. Pyrosequencing 454
Pyrosequencing is based on the generation of light signal through
release of pyrophosphate (PPi) on nucleotide addition.
DNAn + dNTP  DNAn+1 + PPI
PPi is used to generate ATP from adenosine phosphosulfate (APS).
APS + PPI  ATP
ATP and luciferase generate light by conversion of luciferin to
oxyluciferin

21. Pyrosequencing(454 Sequencing)

22. Applied Biosystems:
Ion Torrent PGM

23. Ion Torrent
• Ion Semiconductor Sequencing
• Detection of hydrogen ions during
the polymerization DNA
• Sequencing occurs in microwells
with ion sensors
• No modified nucleotides
• No optics

24. Ion Torrent
• DNA Ions Sequence
– Nucleotides flow sequentially over Ion semiconductor chip
– One sensor per well per sequencing reaction
– Direct detection of natural DNA extension
– Millions of sequencing reactions per chip
– Fast cycle time, real time detection

25. Illumina HiSeq in 2006

27. Platform Updates
Illumina Solexa

30. Rapid Innovation Driving Cost Down

32. OXFORD NANOPORE

34. Sanger vs NGS
• Kilo bp Up to 400 bp
• Need coloning Do not need
• First synthesize then sequencing Seqencing by synthesis

37. References
1. Revolution of DNA Sequencing Method from the Past until
Today(Maryam Alsadat Daneshpour et all )
2. The book: Next Generation Sequencing Data Analysis section II (Xinkun
Wan)
3. .‫پورمشیروهمکاران‬ ‫نادیا‬ ‫توالي‬‫در‬ ‫آن‬ ‫كاربرد‬ ‫و‬ ‫جدید‬ ‫نسل‬ ‫یابي‬‫تشخیص‬ ‫بیماري‬‫هاي‬‫ژنتیك‬
4. Comparison of Next-Generation Sequencing Systems Lin Liu