1. DNA Sequencing -sayed Mohammad Amin Nourion -A’Kia Buford
-Bryanna Menor
-Mariah Nana

2. DNA Sequencing* *DNA sequencing:
Determining the number and order of nucleotides that make up a given molecule of DNA.
Advancement in DNA Sequencing methods have greatly accelerated Bio and Medical research + Discovery

3. Overview
Polymerase Chain Reaction
Shotgun Sequencing
Sanger Method
Next Generation Method

4. Method
Read Length
Sanger
454
Illumina
Ion Torrent

5. Method
Read Length
Sanger bp
454
Illumina
Ion Torrent

6. Method
Read Length
Sanger bp
454 bp
Illumina
Ion Torrent

7. Method
Read Length
Sanger bp
454 bp
Illumina
~100 bp
Ion Torrent

8. Method
Read Length
Sanger bp
454 bp
Illumina
~100 bp
Ion Torrent
~200 bp

9. Polymerase Chain Reaction
Any Target sequence within a DNA sample can be amplified many times to be used in DNA sequencing
Three Step Process
1) Denaturing: Heat briefly to break H-Bonds and separate DNA
2) Annealing: Cooled down to allow primers to attach
3) Extension: DNA Polymerase adds nucleotides to the 3’ end
Done in cycles
Cycle 1 yields 2 DNA molecules
Cycle 2 yields 4 DNA molecules
Cycle 3 yields 8 DNA molecules
After Cycle 3 you have 2 molecules that exactly match the target
Sequence and after 30 cycles you have over 1 Billion matches to the
Target sequence

11. Shotgun Genome Sequencing
Complete genome copies
Fragmented genome chunks

12. Shotgun Genome Sequencing
Fragmented genome chunks
Fragment sizes differ for different seq platforms.

13. Reconstruction 17 bp 66 bp ATTGTTCCCACAGACCG CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
66 bp

14. Reconstruction Final Result:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC

15. Coverage: # of reads underlying the Result
Reconstruction
Final Result:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
Coverage: # of reads underlying the Result

16. Coverage: # of reads underlying the Result
Reconstruction
Final Result:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
6x coverage
100% identity
Coverage: # of reads underlying the Result

17. Coverage: # of reads underlying the Result
Reconstruction
Final Result:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
5x coverage
80% identity
Coverage: # of reads underlying the Result

18. Coverage: # of reads underlying the Result
Reconstruction
Final Result:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
2x coverage
50% identity
Coverage: # of reads underlying the Result

19. Coverage: # of reads underlying the Result
Reconstruction
Final Result:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG
TTGTTCCCACAGACCGTG
TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
1x coverage
Coverage: # of reads underlying the Result

20. Reconstruction

21. Sanger Sequencing Reactions
For a given DNA template, it’s like PCR except:
Uses only a single primer and polymerase to make new single stranded DNA pieces.
Includes regular nucleotides (A, C, G, T) for extension, but also includes dideoxy nucleotides.
Dideoxy Nucleotides A T C G A G T C
Regular Nucleotides
They are Labeled by colour corresponding to base
Are Terminators to a sequence

22. Sanger Sequencing T G C G C G G C C C A5’
T G C G C G G C C C A
Primer
A C G C G C C G G G T ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 5’ 3’

23. Sanger Sequencing T G C G C G G C C C A G T C T T G G G C T5’
T G C G C G G C C C A
Primer
G T C T T G G G C T
A C G C G C C G G G T C A G A A C C C G A T C G C G 5’ 3’

24. Sanger Sequencing T G C G C G G C C C A G T C T T G G G C T A G C G C5’
T G C G C G G C C C A
Primer
G T C T T G G G C T A G C G C
A C G C G C C G G G T C A G A A C C C G A T C G C G 5’ 3’
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp

25. Sanger Sequencing T G C G C G G C C C A G T C T T G G G C T A5’
T G C G C G G C C C A
Primer
G T C T T G G G C T A
A C G C G C C G G G T C A G A A C C C G A T C G C G 5’ 3’
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A G C G C
26 bp

26. Sanger Sequencing T G C G C G G C C C A G5’
T G C G C G G C C C A
Primer G
A C G C G C C G G G T C A G A A C C C G A T C G C G 5’ 3’
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A G C G C
26 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A
22 bp

27. Sanger Sequencing T G C G C G G C C C A G T C T T G G G C5’
T G C G C G G C C C A
Primer
G T C T T G G G C
A C G C G C C G G G T C A G A A C C C G A T C G C G 5’ 3’
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A G C G C
26 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A
22 bp 5’
T G C G C G G C C C A G
12 bp

28. Sanger Sequencing T G C G C G G C C C A G T C T T5’
T G C G C G G C C C A
Primer
G T C T T
A C G C G C C G G G T C A G A A C C C G A T C G C G 5’ 3’
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A G C G C
26 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A
22 bp 5’
T G C G C G G C C C A G
12 bp 5’
T G C G C G G C C C A
G T C T T G G G C
20 bp

29. Sanger Sequencing A C G C G C C G G G T C A G A A C C C G A T C G C G5’ 3’
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A G C G C
26 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A
22 bp 5’
T G C G C G G C C C A G
12 bp 5’
T G C G C G G C C C A
G T C T T G G G C
20 bp 5’
T G C G C G G C C C A
G T C T T
16 bp

30. Sanger Sequencing A C G C G C C G G G T ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?5’ 3’ 5’
T G C G C G G C C C A
? ? ? ? ? ? ? ? ? T
21 bp 5’
T G C G C G G C C C A
? ? ? ? ? ? ? ? ? ? ? ? ? ? C
26 bp 5’
T G C G C G G C C C A
? ? ? ? ? ? ? ? ? ? A
22 bp
Has to be done in a single tube per rxn. 5’
T G C G C G G C C C A G
12 bp 5’
T G C G C G G C C C A
? ? ? ? ? ? ? ? C
20 bp 5’
T G C G C G G C C C A
? ? ? ? T
16 bp

31. Sanger Sequencing T G C G C G G C C C A G T C T G C G C G G C C C A
Laser Reader 5’
T G C G C G G C C C A
G T C
14 bp 5’
T G C G C G G C C C A
G T C T
15 bp 5’
T G C G C G G C C C A
G T C T T G G
18 bp 5’
T G C G C G G C C C A
G T C T T
16 bp 5’
T G C G C G G C C C A
G T C T T G
17 bp 5’
T G C G C G G C C C A
G T
13 bp 5’
T G C G C G G C C C A
G T C T T G G G C T A
22 bp
G T C T T G G G C T 5’
T G C G C G G C C C A
21 bp 5’
T G C G C G G C C C A
G T C T T G G G C
20 bp 5’
T G C G C G G C C C A G
12 bp 5’
T G C G C G G C C C A
G T C T T G G G
19 bp

32. Sanger Sequencing Output
Each sequencing reaction gives us a Spectrogram, usually ~ bp:

33. Sanger Throughput Limitations
Must have 1 colony picked for every 2 reactions
Must do 1 DNA prep for every 2 reactions
Must have 1 PCR tube for each reaction
Must have 1 gel lane for each reaction
from The Economist

34. Shotgun sequencing by Ion Torrent Personal Genome Machine and 454

35. Shotgun sequencing by PGM/454
Genomic
Fragment
Adapters

36. Shotgun sequencing by PGM/454
Genomic
Fragment
Barcode

37. Shotgun sequencing by PGM/454

38. Shotgun sequencing by PGM/454
Bead/ISP
Adapter
Complement
Sequences
The idea is that each bead should be amplified
all over with a SINGLE library fragment.

39. Shotgun sequencing by PGM/454
Problem: How do I do PCR to amplify the fragments without having to use 1 tube for each reaction?

40. Shotgun sequencing by PGM/454

41. Shotgun sequencing by PGM/454

42. Shotgun sequencing by PGM/454

43. Shotgun sequencing by PGM/454

44. Shotgun sequencing by PGM/454

45. Shotgun sequencing by PGM/454

46. Shotgun sequencing by PGM/454

47. Shotgun sequencing by PGM/454

48. Shotgun sequencing by PGM/454

49. Shotgun sequencing by PGM/454

50. Shotgun sequencing by PGM/454
~3.5 µm for Ion Torrent, ~30 µm for 454

51. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: T 5’
T G C G C G G C C C A
Primer
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

52. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: A 5’
T G C G C G G C C C A
Primer
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

53. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: G 5’
T G C G C G G C C C A
Primer G
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

54. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: T 5’
T G C G C G G C C C A
Primer G T
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

55. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: C 5’
T G C G C G G C C C A
Primer G T C
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

56. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: A 5’
T G C G C G G C C C A
Primer G T C
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

57. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: T 5’
T G C G C G G C C C A
Primer G T C
T T
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G

58. Shotgun sequencing by PGM/454
Only give polymerase one nucleotide at a time: G 5’
T G C G C G G C C C A
Primer G T C
T T
G G G
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G
The real power of this method is that it can take place in millions of tiny wells in a single plate at once.

59. Raw 454 data Only give polymerase one nucleotide at a time:5’
T G C G C G G C C C A
Primer G T C
T T
G G G
A C G C G C C G G G T C A G A A C C C G A T C G C G 3’ 5’
If that nucleotide is incorporated, enzymes turn by-products into light:
T C A G T C A G T C A G
The real power of this method is that it can take place in millions of tiny wells in a single plate at once.

60. Ion Torrent Sequencing

61. Ion Torrent Sequencing

62. Final Thoughts
DNA sequencing is becoming vastly faster and more affordable
Generating data is no longer the bottleneck, understanding it is
Bioinformatics types should be in high demand in the near future

63. Comparing Different Technologies
Sanger Sequencing
Advantages
Disadvantages
Lowest error rate
Long read length (~750 bp)
Can target a primer
High cost per base
Long time to generate data
Need for cloning
Amount of data per run

64. Comparing Different Technologies
454 Sequencing
Advantages
Disadvantages
Low error rate
Medium read length
(~ bp)
Relatively high cost per base
Must run at large scale
Medium/high startup costs

65. Comparing Different Technologies
Ion Torrent Sequencing
Advantages
Disadvantages
Low startup costs
Scalable (10 – 1000 Mb of data per run)
Medium/low cost per base
Low error rate
Fast runs (<3 hours)
New, developing technology
Cost not as low as Illumina
Read lengths only ~ bp so far

66. Comparing Different Technologies
Illumina Sequencing
Advantages
Disadvantages
Low error rate
Lowest cost per base
Tons of data
Must run at very large scale
Short read length
(50-75 bp)
Runs take multiple days
High startup costs
De Novo assembly difficult