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DepthOfCoverage Genetics for Dummies 2017 NGS I - History and Technologies Robert Kraaij Department of Internal Medicine [email protected]
DepthOfCoverage Genetics for Dummies 2017
NGS I - History and Technologies
Robert Kraaij
Department of Internal Medicine
Things to be addressed
Sanger sequencing: how it began
NGS: many short reads that might contain errors
Third generation sequencing: now available!
What will NGS bring us?
RFLP
TaqMan
Array
Array and Imputation
Regional Sequencing
Full Genome Sequencing
Overview
• First Generation: Sanger sequencing
• Next (Second) Generation
• Third Generation
1953: Double-Helix Model of DNA
James D. Watson and Francis Crick
from wikipedia.org
4 nucleotides
2 strands
A-T and C-G pairing
1970: HindII the First Restriction Enzyme
Hamilton O. Smith
- T T C G A A - 3’- -5’
- A A G C T T - -3’ 5’-
from wikipedia.org
isolation of clonal DNA fragments
1977: Maxam & Gilbert Sequencing
Walter Gilbert
from wikipedia.org
Maxam & Gilbert Sequencing
G G+A C+T C
1977: Sanger Sequencing
Frederick Sanger
from wikipedia.org
Sanger Sequencing
Sanger Sequencing
Sanger Sequencing
G C A T
Sanger Sequencing
G A T C
Sanger Sequencing
Sanger sequencing landmarks
from wikipedia.org
• 1977 bacteriophage φX174 5.4 kb
• 1984 Epstein-Barr virus 170 kb
• 1995 Haemophilus influenzae 1.8 Mb
• 2001 Human 3 Gb
June 26th, 2000: working draft, 95% gesequenced
April 14th, 2003: finished: 99% gesequenced.
Costs: $ 2.7 billion (instead of $ 3 billion)
Timing: 1990 - 2003 (instead of 2005)
Bill Clinton Tony Blair Craig Venter Francis Collins
The Human Genome Project
Overview
• First Generation: Sanger sequencing
• Next (Second) Generation
• Third Generation
Next Generation: Roche 454
Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
micro-reactors
water-in-oil
emulsion
Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
A
Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
A G C T etc.
Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
A A A T C G G G G G C A
Next Generation: Ion Torrent
Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
A
Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
T G A C etc.
Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
A A A T C G G G G G C A
Next Generation: Illumina
Sequencing Workflow
DNA
isolation
Library
preparation Sequencing
Data
analysis
Sequencing Workflow
DNA
isolation
Library
preparation Sequencing
Data
analysis
Sequencing Workflow
DNA
isolation
Library
preparation Sequencing
Data
analysis
Illumina sequencing
• fragment DNA
• clonal amplification
on flowcell by bridgePCR
• sequencing-by-synthesis
Bridge amplification
Illumina sequencing
• fragment DNA
• clonal amplification
on flowcell by bridgePCR
• sequencing-by-synthesis
Sequencing by synthesis
HP1 primer anneals to adapter
Sequencing by synthesis
A + C + T + G
Sequencing by synthesis
A A A T C G G G G G C A
Sequencing by synthesis
Sequencing by synthesis
Per Cycle Imaging
G A T C
Per Cycle Imaging
MiniSeq MiSeq NextSeq500 HiSeq2500
2 x 150 b 2 x 300 b 2 x 150 b 2 x 125 b
6.6 Gb 13 Gb 100 Gb 450/900 Gb
22M clusters 22M clusters 0.4B clusters 2B/4B clusters
1 day 3 days 1 day 6 days
50k$ 100k€ 250k€ 700k$
4250 $/WG 3500 $/WG
Illumina: Normal flow cell technology
HiSeq4000 HiSeqX Five HiSeqX Ten NovaSeq6000
2 x 150 b 2 x 150 b 2 x 150 b 2 x 150 b
0.65/1.3 Tb 0.8/1.6 Tb 0.8/1.6 Tb 0.85/1.7 Tb
2/4 B clusters 2.5/5 B clusters 2.5/5 B clusters 2.8/5.6 B clusters
4 days 3 days 3 days 2 days
900k$ 5 x 1.2M$ 10 x 1M€ 1M€
2500 $/WG 1500 $/WG 1000 $/WG 1200 $/WG
Illumina: Patterned flow cell technology
Illumina: Patterned flow cell technology
Patterned flowcell
Billions of nanowells
Extreme high density
No overlapping clusters
Special polymerase?
Illumina Whole Genome Sequencing p
rice p
er
wh
ole
gen
om
e (
$)
5,000 -
10,000 -
0 -
price per system
MiSeq
10,000$
NextSeq
4,250$ HiSeq2500
3,500$ HiSeq3000/4000
2,500$
HiSeqX Five
1,500$ HiSeqX Ten
1,000$
Overview
• First Generation: Sanger sequencing
• Next (Second) Generation
• Third Generation
Third generation sequencing =
single molecule sequencing
Third Generation: PacBio
RS
Sequal
• no DNA amplification
• real-time imaging of
DNA polymerase
• sequencing-by-
synthesis
PacBio
SMRT technology
Library prep
Circular DNA
SMRT cell
SMRT technology
>10kb reads
1 Gb output
Better chemistry
De novo assembly
Haplotyping
Variant calling
Posted February 10, 2014
The Genomics Resource Center
University of Maryland
http://www.igs.umaryland.edu
Third Generation: Oxford Nanopore
Oxford Nanopore
Oxford Nanopore
Oxford Nanopore
Library prep
1D or 2D reading
>100kb reads
Not many reads
Oxford Nanopore
6 bases in pore
6x base calling
Caller development
Community
Not ready yet
“Illumina in 2007”
Big improvement 2017
Oxford Nanopore
Things to Remember
Next Generation Sequencing techniques will allow
to interrogate every single base in a genome
Sanger Sequencing is the first generation of
sequencing which is based on chain termination
emulsionPCR is a PCR technique that allows to
perform millions of PCR reactions in one tube
bridgePCR: ditto on a flowcell
NGS: many short reads that contain errors
