1. Sequencing Technologies
2nd Generation
(“NextGen”)
Sequencing Technologies
“Fantastic”
bizarre or exotic; seeming more appropriate to a fairy tale than to reality or practical use

2. Read Length is Not As Important For Resequencing
Jay Shendure

3. Paired End Reads are Important!
Known Distance
Read 1
Read 2
Repetitive DNA
Unique DNA
Paired read maps uniquely
Single read maps to
multiple positions

5. emulsion PCR
emPCR
Margulies M et al., (2006) Genome sequencing in microfabricated high-density picolitre reactors Nature 437,

6. Roche 454
Margulies M et al., (2006) Genome sequencing in microfabricated high-density picolitre reactors Nature 437,

7. OH P P P P P P P
EE Slawson Tempel, © WUSTL

8. OH P P P P P P P
EE Slawson Tempel, © WUSTL

9. OH P P P OH P P P OH P P P OH P P P OH P P P P P P P
EE Slawson Tempel, © WUSTL

10. P P P OH P P P OH P P P P P P P
EE Slawson Tempel, © WUSTL

11. P OH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

12. P
Pyrophosphate OH P P P P P P P P
EE Slawson Tempel, © WUSTL

13. P OH P P P P P P P P
EE Slawson Tempel, © WUSTL

14. ATP
+ luciferin P OH P P P P P P P P
EE Slawson Tempel, © WUSTL

15. ATP
+ luciferin P OH P P P P P P P P
EE Slawson Tempel, © WUSTL

16. P OH P OH P OH P OH OH P P P P P P P P
EE Slawson Tempel, © WUSTL

17. P OH P OH P OH P OH OH P P P P P P P P
EE Slawson Tempel, © WUSTL

18. P OH P OH P OH P OH OH P P P P P P P P
EE Slawson Tempel, © WUSTL

19. P OH P OH P OH P P OH P P P P P P P P
EE Slawson Tempel, © WUSTL

20. P OH P OH P P OH P OH P P P P P P P P
EE Slawson Tempel, © WUSTL

21. P OH P OH P P P P OH P P P P P P P P P
EE Slawson Tempel, © WUSTL

22. P OH P P P OH P OH P P P P P P P P P
EE Slawson Tempel, © WUSTL

23. P OH P P P P OH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

24. P P P P OH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

25. ATP + luciferin P P P P OH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

26. ATP + luciferin P P P P OH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

27. Brightness of flash is proportional to number of nucleotides added
Flash is too bright
4-mer
3-mer
Flash brightness
2-mer
1-mer
TCACTTCAAGGGT…
EE Slawson Tempel, © WUSTL

28. A T G C ~ 0.5 Gb/run Read length 350-400 bp 200 cycles Roche 454
EE Slawson Tempel, © WUSTL

29. Illumina
Nebulizer
~ 400 bp
EE Slawson Tempel, © WUSTL

30. EE Slawson Tempel, © WUSTL

31. EE Slawson Tempel, © WUSTL

32. Flow cell 8 channels (“lanes”)
Surface of flow cell coated with a lawn of oligo pairs

33. EE Slawson Tempel, © WUSTL

34. EE Slawson Tempel, © WUSTL

35. EE Slawson Tempel, © WUSTL

36. EE Slawson Tempel, © WUSTL

37. EE Slawson Tempel, © WUSTL

38. EE Slawson Tempel, © WUSTL

39. EE Slawson Tempel, © WUSTL

40. EE Slawson Tempel, © WUSTL

41. EE Slawson Tempel, © WUSTL

42. EE Slawson Tempel, © WUSTL

43. EE Slawson Tempel, © WUSTL

44. EE Slawson Tempel, © WUSTL

45. EE Slawson Tempel, © WUSTL

46. EE Slawson Tempel, © WUSTL

47. EE Slawson Tempel, © WUSTL

48. EE Slawson Tempel, © WUSTL

49. EE Slawson Tempel, © WUSTL

50. Each piece has a
unique sequence
EE Slawson Tempel, © WUSTL

51. EE Slawson Tempel, © WUSTL

52. EE Slawson Tempel, © WUSTL

53. EE Slawson Tempel, © WUSTL

54. EE Slawson Tempel, © WUSTL

55. EE Slawson Tempel, © WUSTL

56. EE Slawson Tempel, © WUSTL

57. EE Slawson Tempel, © WUSTL

58. EE Slawson Tempel, © WUSTL

59. EE Slawson Tempel, © WUSTL

60. EE Slawson Tempel, © WUSTL

61. EE Slawson Tempel, © WUSTL

62. EE Slawson Tempel, © WUSTL

63. EE Slawson Tempel, © WUSTL

64. EE Slawson Tempel, © WUSTL

65. EE Slawson Tempel, © WUSTL

66. EE Slawson Tempel, © WUSTL

67. EE Slawson Tempel, © WUSTL

68. EE Slawson Tempel, © WUSTL

69. EE Slawson Tempel, © WUSTL

70. EE Slawson Tempel, © WUSTL

71. EE Slawson Tempel, © WUSTL

72. EE Slawson Tempel, © WUSTL

73. EE Slawson Tempel, © WUSTL

74. EE Slawson Tempel, © WUSTL

75. “bridge PCR”
EE Slawson Tempel, © WUSTL

76. thousands of strands/cluster
each cluster (“polony”)
has a unique sequence
EE Slawson Tempel, © WUSTL

77. EE Slawson Tempel, © WUSTL

78. EE Slawson Tempel, © WUSTL

80. EE Slawson Tempel, © WUSTL

81. EE Slawson Tempel, © WUSTL

82. EE Slawson Tempel, © WUSTL

83. EE Slawson Tempel, © WUSTL

84. EE Slawson Tempel, © WUSTL

86. STOP P P P P P P P P P P P P
EE Slawson Tempel, © WUSTL

87. Metzger M (2009) Nature Reviews Genetics 11: 31-46

88. STOP P P P
STOP P P P
STOP P P P
STOP P P P
EE Slawson Tempel, © WUSTL

89. P P P P OH P P P P P P P P P EE Slawson Tempel, © WUSTL STOP STOP STOP

90. P P P P OH P P P P P P P P P EE Slawson Tempel, © WUSTL STOP STOP STOP

91. P
STOP P
STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

92. P
STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

93. STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

94. STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

95. STOP OH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

96. P P P P P P P P OH P P P P P P P P P P EE Slawson Tempel, © WUSTL STOP

97. P P P P P P P P OH P P P P P P P P P P EE Slawson Tempel, © WUSTL STOP

98. P
STOP P
STOP
STOP P P P P P P P P P P P P
EE Slawson Tempel, © WUSTL

99. P
STOP P P P P P P P P P P P
EE Slawson Tempel, © WUSTL

100. STOP P P P P P P P P P P P
EE Slawson Tempel, © WUSTL

101. STOP P P P P P P P P P P P
EE Slawson Tempel, © WUSTL

102. STOP OH P P P P P P P P P P P
EE Slawson Tempel, © WUSTL

103. G…
© Illumina, EEST, © WUSTL

104. GC…
© Illumina, EEST, © WUSTL

105. GCT…
© Illumina, EEST, © WUSTL

106. GCTG…
© Illumina, EEST, © WUSTL

107. GCTGA…
© Illumina, EEST, © WUSTL

108. 100+ Million Clusters
Per Flow Cell
100 Microns

109. Camera time is the limiting step!
Flowcell
8 lanes
For picture taking:
Each lane is broken up into 100 tiles,
each fluor is imaged separately –
2400 pictures taken per cycle
EE Slawson Tempel, © WUSTL

110. Chemistry problem 1: terminator is retained
out of phase
STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

111. Chemistry problem 2: fluor is retainedOH P P P P P P P P P P
EE Slawson Tempel, © WUSTL

112. Chemistry problem 2: fluor is retained
STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

113. Chemistry problem 2: fluor is retained
STOP P P P P P P P P P P
EE Slawson Tempel, © WUSTL

114. Illumina >100 Gb/run HiSeq ~ 3 – 30 Gb/run GAII Read length
90x106 reads/lane * 102 bp/read = 9x109 bp/lane * 16 lanes/run = 144 Gb/run
~ 3 – 30 Gb/run GAII
Read length
30 – 120 bp

115. ABI SOLiD
Support Oligonucleotide Ligation Detection
emPCR

116. ABI SOLiD
Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:

117. ABI SOLiD
Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:

118. ABI SOLID
Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:

119. ABI SOLiD
Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:

120. Mardis ER. (2008) Next-generation DNA sequencing methods
Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:

121. Ion Torrent

122. Nature 475:348 (2011)
~100 bp reads
30 Mb/run 

124. Ion Torrent read quality

125. 454, 7.4X, 24.5 Gb cost < $1M 3.3 million SNPs
10,654 cause aa substitution (7,648 different from Venter)
222,718 indels (2 to 40kb)
18 CNVs (26 kb to 1.6 Mb)
carrier of 10 highly penetrant disease alleles

126. Illumina, 73X, 173 Gb contig N50 = 40 kb scaffold N50 = 1.3 Mb
PMID:
Illumina, 73X, 173 Gb
contig N50 = 40 kb
scaffold N50 = 1.3 Mb

127. ABI SOLiD, 30X coverage 35 interchromosomal translocations
PLoS Genetics 6: e (2010)
ABI SOLiD, 30X coverage
107.5 Gb of raw data
55.51 Gb mapped to genome
35 interchromosomal translocations
1,315 structural variations (>100 bp)
191,743 small (<21 bp) indels
2,384,470 SNVs
512 genes homozygously mutated

128. a recessive EMS-induced mutation affecting egg shell morphology
Genetics : 25–32
a recessive EMS-induced mutation affecting egg shell morphology
Illumina, 8X coverage
103 SNP differences between mutant and wt
9 non-synonomous
2 nonsense >> one in encore, an obvious candidate

129. Illumina 5.1 Gb of sequence 76 bp reads 40X coverage
30 volume 42 | number 1 | january 2010
Illumina
5.1 Gb of sequence
76 bp reads
40X coverage
4 affected individuals

130. RNA-Seq
Pepke S, Wold B & Mortazavi A. (2009) Nature Methods 6:S22

131. ChIP-Seq
Lefrançois P et al. (2009) Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics 10:37

133. Plant Physiology, July 2009, Vol. 150, pp. 1541–1555

134. “Fabulous” 3rd Generation (“Next2Gen”) Sequencing Technologies
having no basis in reality; mythical

135. . A T G C . A T G C . A T G C . A T G C

136. Helicos
Single-molecule
sequencing

137. Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

138. Metzger M (2009) Nature Reviews Genetics 11: 31-46

139. Helicos
105 to 140 Megabases per hour
~ 35 bp average read length

140. (2009) Volume 27: 847
Helicos, 28X coverage, 84 Gb
2.8M SNPs
752 CNVs

141. Ion Torrent
Single-molecule
sequencing

142. Single-molecule sequencing - +
Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

143. Nanopore sequencing - +
Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

144. Nanopore sequencing
Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

145. Pacific Biosciences
Single-molecule
sequencing
Eid et al 2008

146. detection volume 20 zeptoliters (10-21 liters).
emission
excitation
ZMW: a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a silicon dioxide substrate
detection volume 20 zeptoliters (10-21 liters).
PacBio technology backgrounder:

147. PacBio technology backgrounder: http://www. pacificbiosciences

148. When the DNA polymerase encounters the nucleotide complementary to the next base in the template, it is incorporated into the growing DNA chain. During incorporation, the enzyme holds the nucleotide in the ZMWs detection volume for tens of milliseconds, orders of magnitude longer than the average diffusing nucleotide. The system detects this as a flash of bright light because the background is very low. The polymerase advances to the next base and the process continues to repeat
PacBio technology backgrounder:

149. multiple reads of the same molecule
PacBio technology backgrounder:

150. Eid J et al. (2009) Molecules Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323, 133 PMID:

151. Does it work? 150 bp circular template ~93% raw accuracy
15x coverage 99.3% accuracy
Eid et al., 2009

152. ~ 2-5 bp/sec
PacBio claims that, by 2013, the technology will be able to give a
‘raw’ human genome sequence in less than 3 min, and a complete high-quality sequence in 15 min. (
Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

153. F. Sanger, S. Nicklen, and A. R. Coulson, Proc Natl Acad Sci U S A