CG920 Genomics
Lesson 8
Next Generation Sequencing
Roman Hobza
Institute of Biophysics of the Czech Academy of Sciences
hobza@ibp.cz
2
From discovery to technology explosion
 1868: Discovery of DNA
 1953: Watson and Crick propose double helix structure
 1977: Sanger sequencing
 1985: PCR
 2000: Working draft human genome announced (Sanger method)
 2005: 454 sequencer launch (pyrosequencing)
 2006: Genome Analyzer launched (Solexa sequencing)
 2007: SOLiD launched (ligation sequencing)
 2009: Whole human genome no longer merits Nature/Science
paper
 2010: “third-gen” systems
$ human
Genome
$3 billion
$2-3 million
$250k
$50k
$20k
<$1k
2
3
4
5
6
77
7 7
Oxford Nanopore
Sensor array chip: many nanopores in parallel
DNA Sequencing Proteins Polymers Small Molecules
Adaptable protein nanopore:
Application
Specific
GenericPlatform
Electronic read-out system
8
Mechanical damage during tissue homogenization.
Wrong pH and ionic strength of extraction buffer.
Incomplete removal / contamination with nucleases.
Phenol: too old, or inappropriately buffered (pH 7.8 – 8.0); incomplete removal.
Wrong pH of DNA solvent (acidic water).
Recommended: 1:10 TE for short-term storage, or 1xTE for long-term storage.
Vigorous pipetting (wide-bore pipet tips).
Vortexing of DNA in high concentrations.
Too many freeze-thaw cycles (we tested 5, still Ok).
Debatable: sequence-dependent
DNA degradation
9
10
Two strategies
• Whole genome shotgun (bottom-top)
• Clone-by-clone (top-bottom)
Genome sequencing
11
• A rapid progress in next generation sequencing technologies
promises to provide complete (reference) DNA sequences
• The bottleneck:
– NOT the sequencing capacity
– BUT the ability to assemble many short reads with
prevalence of repeated DNA (and polyploidy)
Sequencing without a limit?
12
12
Genome sequencing
GenBank 1982 Los Alamos Sequence Database
Walter Goad
13
Frederick Sanger
1958 – Nobel prize – insuline structure
1975 - Dideoxy sequencing method
1977 – Φ-X174 (5,368 bp) sequence
1980 – second Nobel prize
λ phage sequence
shotgun method (48,502 bp)
14
Genome sequencing
 1986 Leroy Hood:
automatic sequencing machine
 1986 Human Genome Initiative
Leroy Hood
15
Genome sequencing
 1995 John Craig Venter
first bacterial genome
John Craig Venter
16
Craig Venter
Global Ocean Sampling Expedition
Synthetic genomics
Human Longevity Inc
http://www.youtube.com/watch?v=J0rDFbr
hjtI
17
Which applications are labs performing?
17
18
2010 Human genome reference
19
2010 Human genome reference
20
Anne Wojcicki CEO - manželka spoluzakladatele Google
Sergey Mikhaylovich Brin
23andme (30% GSK)
21
22
23
24
25
26
27
28
29
30
 http://www.454.com
Genome Sequencer 20 System
454 pyrosequencing (2005)
31
DNA library preparation
32
Fragmentace DNA
33
Ligace adaptoru
34
Vychytání DNA molekul
35
denaturace
36
37
emPCR
38
Vznik emulze (olej)
39
emPCR
40
emPCR
41
Vychytání kuliček
42
Vychytání kuliček
43
denaturace
44
Sekvenační primer
45
Disperze na sklíčko
46
Disperze na sklíčko
47
Parametry mikroreaktorů
48
Parametry mikroreaktorů
49
sekvenace
50
sekvenace
51
sekvenace
52
sekvenace
53
sekvenace
54
sekvenace
55
sekvenace
56
sekvenace
57
SOLID (Sequencing by Oligonucleotide Ligation and Detection)
2-base encoding sequencing (2007)
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
Solexa (2007)
76
77
78
HELICOS (2008)
True Single Molecule Sequencing (tSMS)
79
Single Molecule Real-Time (SMRT)
Pacific Biosciences
20 zeptolitrů
80
Ion Torrent
81
Oxford nanopore
82
Další technologie
 Mikroelektroforéza
 Sekvenování na bázi microarray
83
CHALLENGES IN GENOME SEQUENCING
De novo genome assemblies using only short read data of NGS
technologies are generally incomplete and highly fragmented due to
 Large duplications
 High proportion of repetitive DNA
- chromosomal approach, BAC-by-BAC sequencing
- challenge!
 Large genome size (~17 Gb)
 Polyploidy (3 subgenomes)
Chromosomal approach
83
84
BAC-BY-BAC SEQUENCING
BAC clones
 Physical map is composed of contigs
of overlapping BAC clones
 BAC contigs are landed on the
chromosome through markers
comprised in the contigs
85
SOLUTIONS FOR THE REPEATS
 Long mate-pair reads > 10 kb
 Long read technologies – PacBio, Oxford Nanopore
 Optical mapping
 Single-molecule mapping of genomic DNA hundreds of kilobases
to several megabases in size
 Creates sequence-motif maps, which provide long-range
template for ordering genomic sequences
 Visualisation of reality “Seeing is Believing”
86
Three enzymatic approaches
 restriction enzymes:
sequence-specifically cleave DNA
immobilized on a surface
 nicking enzymes:
fluorescent labelling
of the nicking site
in solution (BioNano
Genomics - Irys)
 methyltransferase enzymes:
labelling with ultra-high density
OPTICAL MAPPING
Nicking
Strand
displacement
Incorporatio
n of
fluorescent
nucleotides
87
BIONANO GENOME MAPPING ON NANOCHANEL ARRAYS
3 Fluorescence imaging
Lam et al., Nat. Biotechnol. 30(8) 2012
4 Map construction
DNA linearization2
5 Building consensus map
Nickase (Nt.BspQI)
1 Sequence-specific labeling
U U
A
Fluorescent dye conjugated nucleotides (Alexa 546 dUTP) were incorporated at  the
Nt.BspQI sites by Vent (exo−) polymerase. Next, we stained the labeled DNA 
molecules with the
DNA‐intercalating dye, YOYO‐1, which facilitates visualization of the DNA molecule and
measurement of its size. Then, we loaded the DNA onto a nanochannel array chip and
applied an electric field, which gradually drives the long, coiled DNA molecules in free
suspension through a series of micro‐ and nanofluidic structures. Once
the nanochannels were populated by a set of linearized DNA molecules, we imaged 
them with automated high‐resolution fluorescent microscopy. We determined the size 
of each DNA molecule by directly measuring its contour length. The histogram peaks 
represent the location of each sequence motif along the molecules.
87
88
88
Discussion