Handout available at https://drive.google.com/open?id=0B52D4j3rC4WDN2hRV25CaW9kb0E Define DNA sequencing. Describe the history of DNA sequencing. Describe the steps of Sanger sequencing. Define next generation sequencing. Describe the steps of next generation DNA sequencing. Describe use of DNA sequencing.

1. NEXT GENERATION
SEQUENCINGPresented by
Dr. Rayhan Shahrear
MS Resident
Phase A, Year 2, Block 6
Dept. of Anatomy, BSMMU.
Guided by
Prof. Laila Anjuman Banu
Chairman and Professor
Molecular Biology and Genetics
Dept. of Anatomy, BSMMU.

2. Objectives
Audience will be able to-
• define DNA sequencing
• describe the history of DNA sequencing
• describe the steps of Sanger sequencing
• define next generation sequencing
• describe the steps of next generation DNA sequencing
• describe use of DNA sequencing

3. What is DNA sequencing?
Sequencing DNA means determining the order of the four
chemical building blocks - called "bases" - that make up the
DNA molecule.

4. History of DNA Sequencing
• 1953 - Watson and Crick
• 1965 - Robert Holley
• 1970 - Ray WU
• 1977 - Fredric Sanger and Maxam-Gilbert
• 1986 - Semi-automated DNA sequencing machine
• 1990 - Step by step sequencing
• 1996 - Pyrosequencing
• 1997 - DNA colony sequencing
• 1998 - Phred quality score
• 2000 - Massively parallel signature sequencing
• 2004 - Massively parallel pyrosequencing

5. SANGER SEQUENCING

6. 1977

9. dNTPs +
Primers +
Enzymes
ddNTPs
ddATPs ddTTPs ddCTPs ddGTPs

11. T A G A T G C T G
ddATPs
ddTTPs
ddCTPs
ddGTPs

12. NEXT GENERATION
SEQUENCING

13. What is NGS?
Next-generation sequencing (NGS), also known as high-
throughput sequencing is the catch-all term used to describe
a number of different modern sequencing technologies.
E.g.
• Pyrosequencing
• Illumina sequencing
• SOLiD sequencing
• Ion Torrent semiconductor sequencing etc.

14. Illumina sequencing

15. Steps
• Creating DNA libraries
• Tagmentation
• Indexing
• Cleaning up
• Normalization
• Sequence by synthesis
• Loading flow cell
• Amplification
• Sequencing
• Analysis of the sequenced data

16. But…

17. DNA Extraction

18. Quality and Quantity Check

19. PCR

20. Amplicon Quantity Check

22. Creating DNA library
CleavingTaggingIndexing

23. A 1
B 1
C 1
D 1
E 1
F 1
G 1
H 1
A 2
B 2
C 2
D 2
E 2
F 2
G 2
H 2
A 3
B 3
C 3
D 3
E 3
F 3
G 3
H 3
A 4
B 4
C 4
D 4
E 4
F 4
G 4
H 4
A 5
B 5
C 5
D 5
E 5
F 5
G 5
H 5
A 6
B 6
C 6
D 6
E 6
F 6
G 6
H 6
A 7
B 7
C 7
D 7
E 7
F 7
G 7
H 7
A 8
B 8
C 8
D 8
E 8
F 8
G 8
H 8
A 9
B 9
C 9
D 9
E 9
F 9
G 9
H 9
A 10
B 10
C 10
D 10
E 10
F 10
G 10
H 10
A 11
B 11
C 11
D 11
E 11
F 11
G 11
H 11
A 12
B 12
C 12
D 12
E 12
F 12
G 12
H 12
9 10 11 1287654321
A
B
C
D
E
F
G
H

24. Sequence by Synthesis
Loading flow cell

25. Sequence by Synthesis
Amplification

26. Sequence by Synthesis
Amplification

27. Sequence by Synthesis
Sequencing

30. Data Analysis

31. Use of DNA Sequencing
• It helps to understand and comprehend the internal
structure of genes in the DNA.
• It helps to understand which sequence codes for what kind
of proteins.
• The knowledge of sequence can tell if there is any disease
or not.
• The knowledge of sequence can be used to prepare
proteins.
• The knowledge of sequencing will help to cure many
diseases.

32. Why NGS?
• Sequencing mechanism
• Read length
• Accuracy
• Reads
• Output data/run
• Time/run
• Advantage
• Disadvantage
• Instrument price
• Library preparation
• Cost

33. NGS Sanger sequencing
Sequencing mechanism Sequencing by synthesis Dideoxy chain termination
Read length 50SE, 50PE, 101PE 400 ∼ 900 bp
Accuracy 98%, (100PE) 99.999%
Reads 30k Whole
Output data/run 600 Gb 1.9~84 Kb
Time/run 3~10 Days 20 Mins~3 Hours
Advantage High throughput High quality, long read length
Disadvantage Short read assembly High cost low throughput
Instrument price
Instrument $690,000,
human genome
Instrument $95,000, about $4
800 bp reaction
Library preparation Yes No
Cost/million bases $0.07 $2400

34. Thank You