depthofcoverage genetics for dummies 2017 slides r… · depthofcoveragegenetics for dummies 2017...
TRANSCRIPT
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DepthOfCoverage Genetics for Dummies 2017
NGS I - History and Technologies
Robert Kraaij
Department of Internal Medicine
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Things to be addressed
Sanger sequencing: how it began
NGS: many short reads that might contain errors
Third generation sequencing: now available!
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What will NGS bring us?
RFLP
TaqMan
Array
Array and Imputation
Regional Sequencing
Full Genome Sequencing
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Overview
• First Generation: Sanger sequencing
• Next (Second) Generation
• Third Generation
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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
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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
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1977: Maxam & Gilbert Sequencing
Walter Gilbert
from wikipedia.org
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Maxam & Gilbert Sequencing
G G+A C+T C
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1977: Sanger Sequencing
Frederick Sanger
from wikipedia.org
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Sanger Sequencing
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Sanger Sequencing
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Sanger Sequencing
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G C A T
Sanger Sequencing
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G A T C
Sanger Sequencing
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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
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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
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Overview
• First Generation: Sanger sequencing
• Next (Second) Generation
• Third Generation
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Next Generation: Roche 454
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Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
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Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
micro-reactors
water-in-oil
emulsion
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Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
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Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
A
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Roche 454
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads in
PicoTiterPlate
• sequencing-by-
synthesis
A G C T etc.
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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
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Next Generation: Ion Torrent
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Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
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Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
A
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Ion Torrent
• fragment DNA
• clonal amplification
on bead by emPCR
• load beads on chip
• sequencing-by-
synthesis
T G A C etc.
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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
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Next Generation: Illumina
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Sequencing Workflow
DNA
isolation
Library
preparation Sequencing
Data
analysis
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Sequencing Workflow
DNA
isolation
Library
preparation Sequencing
Data
analysis
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Sequencing Workflow
DNA
isolation
Library
preparation Sequencing
Data
analysis
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Illumina sequencing
• fragment DNA
• clonal amplification
on flowcell by bridgePCR
• sequencing-by-synthesis
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Bridge amplification
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Illumina sequencing
• fragment DNA
• clonal amplification
on flowcell by bridgePCR
• sequencing-by-synthesis
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Sequencing by synthesis
HP1 primer anneals to adapter
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Sequencing by synthesis
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A + C + T + G
Sequencing by synthesis
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A A A T C G G G G G C A
Sequencing by synthesis
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Sequencing by synthesis
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Per Cycle Imaging
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G A T C
Per Cycle Imaging
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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 $/WG
Illumina: Normal flow cell technology
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HiSeq4000 HiSeqX Five HiSeqX Ten NovaSeq6000
2 x 150 b 2 x 150 b 2 x 150 b 2 x 150 b
0.65/1.3 Tb 0.8/1.6 Tb 0.8/1.6 Tb 0.85/1.7 Tb
2/4 B clusters 2.5/5 B clusters 2.5/5 B clusters 2.8/5.6 B clusters
4 days 3 days 3 days 2 days
900k$ 5 x 1.2M$ 10 x 1M€ 1M€
2500 $/WG 1500 $/WG 1000 $/WG 1200 $/WG
Illumina: Patterned flow cell technology
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Illumina: Patterned flow cell technology
Patterned flowcell
Billions of nanowells
Extreme high density
No overlapping clusters
Special polymerase?
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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$
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Overview
• First Generation: Sanger sequencing
• Next (Second) Generation
• Third Generation
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Third generation sequencing =
single molecule sequencing
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Third Generation: PacBio
RS
Sequal
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• no DNA amplification
• real-time imaging of
DNA polymerase
• sequencing-by-
synthesis
PacBio
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SMRT technology
Library prep
Circular DNA
SMRT cell
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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
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Third Generation: Oxford Nanopore
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Oxford Nanopore
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Oxford Nanopore
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Oxford Nanopore
Library prep
1D or 2D reading
>100kb reads
Not many reads
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Oxford Nanopore
6 bases in pore
6x base calling
Caller development
Community
Not ready yet
“Illumina in 2007”
Big improvement 2017
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Oxford Nanopore
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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