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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
!
Evolution of Sequencing
!
Workshop in Applied Phylogenetics
March 9, 2014
Bodega Bay Marine Lab
!
Jonathan A. Eisen
UC Davis Genome Center
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Review Papers
Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303. doi: 10.1146/annurev-
anchem-062012-092628.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Review Papers
Next-Generation DNA
Sequencing Methods
Elaine R. Mardis
Departments of Genetics and Molecular Microbiology and Genome Sequencing Center,
Washington University School of Medicine, St. Louis MO 63108; email: emardis@wustl.edu
k links to
ontent online,
his volume
articles
ve search
ther
Annu. Rev. Genomics Hum. Genet. 2008.
9:387–402
First published online as a Review in Advance on
June 24, 2008
The Annual Review of Genomics and Human Genetics
is online at genom.annualreviews.org
Annu.Rev.Genom.HumanGenet.2008.9:
byUniversidadNacionalAutonom
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Open Access Papers of Interest
• http://www.microbialinformaticsj.com/content/2/1/3/
• http://www.hindawi.com/journals/bmri/2012/251364/abs/
• http://m.cancerpreventionresearch.aacrjournals.org/
content/5/7/887.full
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Approaching to NGS
Discovery of DNA structure
(Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31)
1953
Sanger sequencing method by F. Sanger
(PNAS ,1977, 74: 560-564)
1977
PCR by K. Mullis
(Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73)
1983
Development of pyrosequencing
(Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365)
1993
1980
1990
2000
2010
Single molecule emulsion PCR 1998
Human Genome Project
(Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351)
Founded 454 Life Science 2000
454 GS20 sequencer
(First NGS sequencer)
2005
Founded Solexa 1998
Solexa Genome Analyzer
(First short-read NGS sequencer)
2006
GS FLX sequencer
(NGS with 400-500 bp read lenght)
2008
Hi-Seq2000
(200Gbp per Flow Cell)
2010
Illumina acquires Solexa
(Illumina enters the NGS business)
2006
ABI SOLiD
(Short-read sequencer based upon ligation)
2007
Roche acquires 454 Life Sciences
(Roche enters the NGS business)
2007
NGS Human Genome sequencing
(First Human Genome sequencing based upon NGS technology)
2008
Miseq
Roche Jr
Ion Torrent
PacBio
Oxford
From Slideshare presentation of Cosentino Cristian
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Sequencing Technology Timeline
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Generation I: Manual Sequencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Maxam-Gilbert Sequencing
http://www.pnas.org/content/74/2/560.full.pdf
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Sanger Sequencing of PhiX174
http://www.ncbi.nlm.nih.gov/
pmc/articles/PMC431765/
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Sanger Sequencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Sanger Sequencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Nobel Prize 1980: Berg, Gilbert, Sanger
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Generation II: Automated Sanger
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Automation of Sanger Part I
Sanger method with labeled dNTPs
The Sanger mehtods is based on the idea that inhibitors can
terminate elongation of DNA at specific points
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Automation of Sanger Part II
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Automated Sanger Highlights
• 1991: ESTs by Venter
• 1995: Haemophilus influenzae genome
• 1996: Yeast, archaeal genomes
• 1999: Drosophila genome
• 2000: Arabidopsis genome
• 2000: Human genome
• 2004: Shotgun metagenomics
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Generation III: Clusters not Clones
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Generation III = “NextGen”
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
NextGen Sequencing OutlineNext-generation sequencing platforms
Isolation and purification of
target DNA
Sample preparation
Library validation
Cluster generation
on solid-phase
Emulsion PCR
Sequencing by synthesis
with  3’-blocked reversible
terminators
Pyrosequencing Sequencing by ligation
Single colour imaging
Sequencing by synthesis
with  3’-unblocked reversible
terminators
AmplificationSequencingImaging
Four colour imaging
Data analysis
Roche 454Illumina GAII ABi SOLiD Helicos HeliScope
From Slideshare presentation of
Cosentino Cristian
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high-throughput-equencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Pyrosequencing
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
Illumina GAII
From Slideshare presentation of
Cosentino Cristian
http://www.slideshare.net/cosentia/high-
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NextGen #1: 454
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Pyrosequencing
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
Illumina GAII
NextGen #1: Roche 454
From Slideshare presentation of
Cosentino Cristian
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Pyrosequencing
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
Illumina GAII
NextGen #1: Roche 454
From Slideshare presentation of
Cosentino Cristian
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Roche 454 Wokflow
From http://acb.qfab.org/acb/ws09/presentations/Day1_DMiller.pdf
http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
a
b
DNA library preparation
Emulsion PCR
A
A
A B
B
B
4.5 hours
8 hours
Ligation
Selection
(isolate AB
fragments
only)
•Genome fragmented
by nebulization
•No cloning; no colony
picking
•sstDNA library created
with adaptors
•A/B fragments selected
using avidin-biotin
purification
gDNA sstDNA library
gDNA
fragmented by
nebulization or
sonication
Fragments are end-
repaired and ligated to
adaptors containing
universal priming sites
Fragments are denatured and
AB ssDNA are selected by
avidin/biotin purification
(ssDNA library)
From Mardis 2008. Annual Rev. Genetics 9: 387.
Roche 454 Step 1: Libraries
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Anneal sstDNA to an excess of
DNA capture beads
Emulsify beads and PCR
reagents in water-in-oil
microreactors
Clonal amplification occurs
inside microreactors
Break microreactors and
enrich for DNA-positive
beads
b
c
Emulsion PCR
Sequencing
A
A B
B
8 hours
7.5 hours
only) with adaptors
•A/B fragments selected
using avidin-biotin
purification
gDNA sstDNA library
sstDNA library Bead-amplified sstDNA library
From Mardis 2008. Annual Rev. Genetics 9: 387.
Roche 454 Step 2: Emulsion PCR
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Anneal sstDNA to an excess of
DNA capture beads
Emulsify beads and PCR
reagents in water-in-oil
microreactors
Clonal amplification occurs
inside microreactors
Break microreactors and
enrich for DNA-positive
beads
Amplified sstDNA library beads Quality filtered bases
c
Sequencing
7.5 hours
sstDNA library Bead-amplified sstDNA library
•Well diameter: average of 44 µm
•400,000 reads obtained in parallel
•A single cloned amplified sstDNA
bead is deposited per well
390 Mardis
From Mardis 2008. Annual Rev. Genetics 9: 387.
Roche 454 Step 3: Pyrosequencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Pyrosequencing
44 µm
Pyrosequecning
Reads are recorded as flowgrams
Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402
Nature Reviews genetics, 2010, 11: 31-46
Sanger
method
-
ABi SOLiD
HeliScope
Nanopore
Roche 454
Illumina GAII
From Slideshare
presentation of Cosentino
Cristian
http://www.slideshare.net/
cosentia/high-throughput-
equencing
Roche 454 Step 3: Pyrosequencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Roche 454 Key Issues
• Number of repeated nucleotides
estimated by amount of light ... many
errors
• Reasonable number of failures in EM-
PCR and other steps
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Roche 454 Evolution
http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
NextGen #2: Solexa
Sequecning by synthesis with reversible terminator
From Slideshare
presentation of
Cosentino Cristian
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www.slideshare.net/
cosentia/high-
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equencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Sequecning by synthesis with reversible terminator
From Slideshare
presentation of
Cosentino Cristian
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NextGen #2: Solexa Illumina
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
NextGen #2: Illumina Accessories
Cluster station
Genome Analyzer IIxPaired-end module Linux server
Bioanalyzer 2100
Instrumentation
le
tion
ers
ation
ng by
esis
sis
ne
ction
GAII
h
hput
From Slideshare
presentation of
Cosentino
Cristian
http://
www.slideshare.
net/cosentia/
high-throughput-
equencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Illumina Outline
Clusters
amplification
Clusterstation
Wash cluster
station
Clustergeneration
Linearization,
Blocking and
primer
Hybridization
Read 1
Prepare read 2
Read 2
GAIIx&PE
SBSsequencing
Pipeline base call
Data analysis
Sample
preparation and
library validation
Analysis
Sequencing workflow
Sample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
From Slideshare
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Illumina Step 1: Prep & Attach DNA
continues for a specific number of cycles, as de-
termined by user-defined instrument settings,
which permits discrete read lengths of 25–35
read and a quality checking pipeline evaluates
the Illumina data from each run, removing
poor-quality sequences.
Adapter
DNA fragment
Dense lawn
of primers
Adapter
Attached
DNA
Adapters
Prepare genomic DNA sample
Randomly fragment genomic DNA
and ligate adapters to both ends of
the fragments.
Attach DNA to surface
Bind single-stranded fragments
randomly to the inside surface
of the flow cell channels.
Nucleotides
a
From Mardis 2008. Annual Rev. Genetics 9: 387.
Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a
nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are
isolated via gel extraction and amplified using limited cycles of PCR
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Illumina Step 2: Clusters by Bridge PCR
Attached
Prepare genomic DNA sample
Randomly fragment genomic DNA
and ligate adapters to both ends of
the fragments.
Attach DNA to surface
Bind single-stranded fragments
randomly to the inside surface
of the flow cell channels.
Bridge amplification
Add unlabeled nucleotides
and enzyme to initiate solid-
phase bridge amplification.
Denature the double
stranded molecules
Nucleotides
Figure 2
The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and all four fluorescently
labeled, 3′-OH blocked nucleotides are added to the flow cell with DNA polymerase. The cluster strands are extended by one
nucleotide. Following the incorporation step, the unused nucleotides and DNA polymerase molecules are washed away, a scan buffer is
added to the flow cell, and the optics system scans each lane of the flow cell by imaging units called tiles. Once imaging is completed,
chemicals that effect cleavage of the fluorescent labels and the 3′-OH blocking groups are added to the flow cell, which prepares the
cluster strands for another round of fluorescent nucleotide incorporation.
392 Mardis
From Mardis 2008. Annual Rev. Genetics 9: 387.
From : http://seqanswers.com/forums/showthread.php?t=21. Steps 2-6: Cluster Generation by Bridge Amplification. In contrast to the 454 and ABI methods which
use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow
cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample
preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based
extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and
extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is
referred to as Illumina's "cluster station", an automated flow cell processor.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Clusters
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
tween 2–4 Gb of DNA sequence data. Once different from that already established for
b
Laser
First chemistry cycle:
determine first base
To initiate the first
sequencing cycle, add
all four labeled reversible
terminators, primers, and
DNA polymerase enzyme
to the flow cell.
Image of first chemistry cycle
After laser excitation, capture the image
of emitted fluorescence from each
cluster on the flow cell. Record the
identity of the first base for each cluster.
Sequence read over multiple chemistry cycles
Repeat cycles of sequencing to determine the sequence
of bases in a given fragment a single base at a time.
Before initiating the
next chemistry cycle
The blocked 3' terminus
and the fluorophore
from each incorporated
base are removed.
GCTGA...
From Mardis 2008. Annual Rev. Genetics 9: 387.
Illumina Step 3: Sequencing by Synthesis
From : http://seqanswers.com/forums/showthread.php?t=21. Steps 7-12: Sequencing by Synthesis. A flow cell containing millions of unique clusters is
now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single
fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal
above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated
at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster.
Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
SBS technology
Sample
reparation
Clusters
mplification
quencing by
synthesis
Analysis
pipeline
troduction
umina GAII
High
hroughput
From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/
cosentia/high-
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Illumina Step 3: Sequencing by Synthesis
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Laser
DNA polymerase enzyme
to the flow cell.
Image of first chemistry cycle
After laser excitation, capture the image
of emitted fluorescence from each
cluster on the flow cell. Record the
identity of the first base for each cluster.
Sequence read over multiple chemistry cycles
Repeat cycles of sequencing to determine the sequence
of bases in a given fragment a single base at a time.
Before initiating
next chemistry cy
The blocked 3' termi
and the fluorophore
from each incorpora
base are removed.
GCTGA...
Figure 2
(Continued )
www.annualreviews.org • Next-Generation DNA Sequencing Methods 393
From Mardis 2008. Annual Rev. Genetics 9: 387.
Illumina Step 3: Cycling
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Illumina Evolution
http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
MiSeq Dx
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
HiSeq x Ten
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
HiSeq x Ten
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
NextGen #3: 454: ABI SolidSequecning by ligation
Sanger
method
Roche 454
-
HeliScope
Nanopore
ABi SOLiD
Illumina GAII
From Slideshare
presentation of
Cosentino Cristian
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
ABI Solid Details
G09-20 ARI 25 July 2008 14:57
A C G T
1stbase
2nd base
A
C
G
T
3'TAnnnzzz5'
3'TCnnnzzz5'
3'TGnnnzzz5'
3'TTnnnzzz5'
Cleavage site
Di base probesSOLiD™ substrate
3'
TA
AT
Universal seq primer (n)
3'
P1 adapter Template sequence
POH
Universal seq primer (n–1)
Ligase
Phosphatase
+
1. Prime and ligate
2. Image
4. Cleave off fluor
5. Repeat steps 1–4 to extend sequence
3'
Universal seq primer (n–1)
1. Melt off extended
sequence
2. Primer reset3'
AA AC G
G GG
C C
C
T AA
A GG
CC
T TTT
6. Primer reset
7. Repeat steps 1–5 with new primer
8. Repeat Reset with , n–2, n–3, n–4 primers
TA
AT
AT
3'
TA
AT
3'
Excite Fluorescence
Cleavage agent
P
HO
TA
AA AG AC AAAT
TT TC TG TT AC
TG
CG
GC
3'
3. Cap unextended strands
3'
PO4
1 2 3 4 5 6 7 ... (n cycles)Ligation cycle
3'
3'1 μm
bead
1 μm
bead
1 μm
bead
–1
Universal seq primer (n)1
Primer round 1
Template
Primer round 2 1 base shift
Glass slide
3'5' Template sequence
1 μm
bead P1 adapter
Read position 35343332313029282726252423222120191817161514131211109876543210
a
The ligase-mediated sequencing approach of the
Applied Biosystems SOLiD sequencer. In a
manner similar to Roche/454 emulsion PCR
amplification, DNA fragments for SOLiD
sequencing are amplified on the surfaces of 1-μm
magnetic beads to provide sufficient signal during
the sequencing reactions, and are then deposited
onto a flow cell slide. Ligase-mediated
sequencing begins by annealing a primer to the
shared adapter sequences on each amplified
fragment, and then DNA ligase is provided along
with specific fluorescent- labeled 8mers, whose
4th and 5th bases are encoded by the attached
fluorescent group. Each ligation step is followed
by fluorescence detection, after which a
regeneration step removes bases from the
ligated 8mer (including the fluorescent group)
and concomitantly prepares the extended primer
for another round of ligation. (b) Principles of two-
base encoding. Because each fluorescent group
on a ligated 8mer identifies a two-base
combination, the resulting sequence reads can
be screened for base-calling errors versus true
polymorphisms versus single base deletions by
aligning the individual reads to a known high-
quality reference sequence.
From Mardis 2008. Annual Rev.
Genetics 9: 387.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
ABI Solid Evolution
http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Complete Genomics
REVIEW
ased the number of false positive gene
ely reduced the number gene candidates
sitosterolemia phenotype were determined after comparison of
the patient’s genome to a collection of reference genomes.
omplete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DNB
f the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a set
onding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. Reprinted with
pyright XXXX American Association for the Advancement of Science.
gure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DN
nthesis, and dimensions of the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a
Figure 3. Schematic of Complete Genomics’
DNB array generation and cPAL
technology. (A) Design of sequencing
fragments, subsequent DNB synthesis, and
dimensions of the patterned nanoarray
used to localize DNBs illustrate the DNB
array formation. (B) Illustration of
sequencing with a set of common probes
corresponding to 5 bases from the distinct
adapter site. Both standard and extended
anchor schemes are shown.
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Comparison in 2008
Roche (454) Illumina SOLiD
Chemistry Pyrosequencing Polymerase-based Ligation-based
Amplification Emulsion PCR Bridge Amp Emulsion PCR
Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb
Mb/run 100 Mb 1300 Mb 3000 Mb
Time/run 7 h 4 days 5 days
Read length 250 bp 32-40 bp 35 bp
Cost per run
(total)
$8439 $8950 $17447
Cost per Mb $84.39 $5.97 $5.81
From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/
CSHL_nextgen.ppt
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Comparison in 2012
Roche (454) Illumina SOLiD
Chemistry Pyrosequencing Polymerase-based Ligation-based
Amplification Emulsion PCR Bridge Amp Emulsion PCR
Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb
Mb/run 100 Mb 1300 Mb 3000 Mb
Time/run 7 h 4 days 5 days
Read length 250 bp 32-40 bp 35 bp
Cost per run
(total)
$8439 $8950 $17447
Cost per Mb $84.39 $5.97 $5.81
From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/
CSHL_nextgen.ppt
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Bells and Whistles
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Multiplexing
From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Multiplexing
http://res.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Small Amounts of DNA
http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Capture MethodsHigh throughput sample preparation
Sample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
Nature Methods, 2010, 7: 111-118
RainDance
Microdroplet PCR
Roche Nimblegen
Salid-phase capture with custom-
designed oligonucleotide microarray
Reported 84% of
capture efficiency
Reported 65-90% of capture efficiency
From Slideshare presentation of Cosentino Cristian
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
High throughput sample preparation
Sample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
Agilent SureSelect
Solution-phase capture with
streptavidin-coated magnetic beads
Reported 60-80% of capture efficiency
From Slideshare presentation of
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high-throughput-equencing
Capture Methods
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Illumina Paired Ends
Paired-end technology
Paired-end sequencing works into GA and uses chemicals from the PE
module to perform cluster amplification of the reverse strandSample
preparation
Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
Introduction
Illumina GAII
High
throughput
From Slideshare
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Moleculo
Large fragments
DNA
Isolate and amplify
CACC GGAA TCTC ACGT AAGG GATC AAAA
Sublibrary w/ unique barcodes
Sequence w/ Illumina
Assemble seqs w/ same codes
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Generation III+: Faster w/ Clusters
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Ion Torrent PGM
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Applied Biosystems Ion Torrent PGM
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Applied Biosystems Ion Torrent PGM
Workflow similar to
that for Roche/454
systems.
!
Not surprising,
since invented by
people from 454.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Ion Torrent pH Based Sequencing
Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Ion Torrent Evolution
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Generation IV: Single Molecule
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Single Molecule I: Helicos
3rd
Generation Sequencing
by
low for
lecular
scent_sequencing
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Single Molecule II: Pacific Biosciences
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Single Molecule II: Pacific Biosciences
Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Analytical Chemistry REVIEW
Φ29 polymerase. Each amplified product of a circularized
fragment is called a DNA nanoball (DNB). DNBs are selectively
attached to a hexamethyldisilizane (HMDS) coated silicon chip
that is photolithographically patterned with aminosilane active
sites. Figure 3A illustrates the DNB array design.
The use of the DNBs coupled with the highly patterned array
offers several advantages. The production of DNBs increases
signal intensity by simply increasing the number of hybridization
sites available for probing. Also, the size of the DNB is on the
same length scale as the active site or “sticky” spot patterned on
Each hybridization and ligation cycle is followed by fluorescent
imaging of the DNB spotted chip and subsequently regeneration
of the DNBs with a formamide solution. This cycle is repeated
until the entire combinatorial library of probes and anchors is
examined. This formula of the use of unchained reads and
regeneration of the sequencing fragment reduces reagent con-
sumption and eliminates potential accumulation errors that can
arise in other sequencing technologies that require close to
completion of each sequencing reaction.19,52,53
Complete Genomics showcased their DNB array and cPAL
Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single DNA
polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at the bottom
surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing template. The corresponding
temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright
2009 American Association for the Advancement of Science.
Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single
DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at
the bottom surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing
template. The corresponding temporal fluorescence detection with respect to each of the five incorporation steps is shown below.
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
Single Molecule II: Pacific Biosciences
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Why Finish Genomes?
The Value of Finished Bacterial Genomes
Why Are Finished Genomes So Important?
When Sanger sequencing was the only available sequencing technique, it was expensive — but not unusual — to
improve genome drafts until they were good enough to be considered finished. With the availability of short-read
sequencing technologies, draft genomes became cheap and easy to produce, and the majority of researchers
skipped the more labor- and time-intensive task of finishing genomes, with the realization that critical data
may be missing (Figure 3). Finished genomes are crucial for understanding microbes and advancing the field of
microbiology3
because:
• Functional genomic studies demand a high-quality,
complete genome sequence as a starting point
• Comparative genomics is meaningful only in terms
of complete genome sequences
• Understanding genome organization provides
biological insights
• Microbial forensics requires at least one complete
reference genome sequence
• Finished genomes aid in microbial outbreak source
identification and phylogenetic analysis
• A complete genome is a permanent scientific
resource
Figure 3: History of drafted vs. finished genomes (adapted from ref. 2).
Microbial Genetics Using SMRT Sequencing
0
2000
4000
6000
8000
10000
12000
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Numberofgenomes
Drafted Bacterial Genomes
Finished Bacterial Genomes
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Why Finish Genomes
JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 6403–6405 Vol. 184, No. 23
0021-9193/02/$04.00ϩ0 DOI: 10.1128/JB.184.23.6403–6405.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
DIALOG
The Value of Complete Microbial Genome Sequencing
(You Get What You Pay For)
Claire M. Fraser,* Jonathan A. Eisen, Karen E. Nelson, Ian T. Paulsen,
and Steven L. Salzberg
The Institute for Genomic Research, Rockville, Maryland 20850
Since the publication of the complete Haemophilus influen-
zae genome sequence in July 1995 (4), the field of microbiology
has been one of the largest beneficiaries of the breakthroughs
in genomics and computational biology that made this accom-
plishment possible. When the 1.8-Mbp H. influenzae project
began in 1994, it was not certain that the whole-genome shot-
gun sequencing strategy would succeed because it had never
been attempted on any piece of DNA larger than an average
lambda clone (ϳ40 kbp) (9).
During the past 7 years, progress in DNA sequencing tech-
nology, the design of new vectors for library construction for
use in shotgun sequencing projects, significant improvements
in closure and finishing strategies, and more sophisticated and
robust methods for gene finding and annotation have dramat-
ically reduced the time required for each stage of a genome
organisms to be sampled because of the cost savings that would
come from not taking each project to completion. While this
strategy does achieve a cost savings, today it is only approxi-
mately 50%, and this comes at a cost in terms of the quality and
utility of the finished product.
A complete genome sequence represents a finished product
in which the order and accuracy of every base pair have been
verified. In contrast, a draft sequence, even one of high cov-
erage, represents a collection of contigs of various sizes, with
unknown order and orientation, that contain sequencing errors
and possible misassemblies. As stated by Selkov et al. in a 2000
paper on a draft sequence of Thiobacillus ferrooxidans, “It is
clear that such sequencing. . .produces more errors than com-
plete genome sequencing. . . . The current error rate is esti-
mated to be 1 per 1,000 to 2,000 base pairs vs. 1 in 10,000 base
http://jb.asm.orDownloadedfrom
http://jb.asm.org/content/184/23/6403.full
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
HGAP Assembly from PacBio
PacBio assembly CDC assembly
Illumina assemblySanger validation
HGAP Assembler for PacBio Data
http://www.pacificbiosciences.com/pdf/
microbial_primer.pdf
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Detecting Modified Bases
Page 2 www.pacb.com/basemod
processes.
The potential benefits of detecting base modification,
using SMRT sequencing, include:
Single-base resolution detection of a wide
the presence of a modified base in the DNA
template
3
. This is observable as an increased space
between fluorescence pulses, which is called the
interpulse duration (IPD), as shown in Figure 2.
Figure 2. Principle of detecting modified DNA bases during SMRT sequencing. The presence of the modified base in the DNA
template (top), shown here for 6-mA, results in a delayed incorporation of the corresponding T nucleotide, i.e. longer
interpulse duration (IPD), compared to a control DNA template lacking the modification (bottom).
3
http://www.pacificbiosciences.com/pdf/microbial_primer.pdf
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this
membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. By measuring that current it is possible to identify the
molecule in question. For example, this system can be used to distinguish the four standard DNA bases and G, A, T and C, and also modified bases. It can be used to identify
target proteins, small molecules, or to gain rich molecular information for example to distinguish the enantiomers of ibuprofen or molecular binding dynamics.
From Oxford Nanopores Web Site
Single Molecule III: Oxford Nanopores
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
• Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore.
(A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue
139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the
cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides
from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL
protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand
sequencing: ssDNA is threaded through a protein nanopore and individual bases are identified, as the strand remains intact.
Panels A, B, and D reprinted with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted
with permission from Oxford Nanopore Technologies (Zoe McDougall).	

tical Chemistry RE
6. Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of RHL protein nanopore mutant depicting the position
extrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the loca
inines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleav
tides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein na
ows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded th
n nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Co
Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall).
Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore.(A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue
139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is
attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein
nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases ar
identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford
Nanopore Technologies (Zoe McDougall).
Single Molecule III: Oxford Nanopores
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Analytical Chemistry REVIEW
would result, in theory, in detectably altered current flow through
he pore. Theoretically, nanopores could also be designed to
measure tunneling current across the pore as bases, each with a
istinct tunneling potential, could be read. The nanopore ap-
lipid bilayer, using ionic current blockage method. The author
predicted that single nucleotides could be discriminated as lon
as: (1) each nucleotide produces a unique signal signature; (2
the nanopore possesses proper aperture geometry to accommo
igure 5. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A) In electronic nanopore schemes
ignal is obtained through ionic current,73
tunneling current,78
and voltage difference79
measurements. Each method must produce a characteristic signa
o differentiate the four DNA bases. Reprinted with permission from ref 83. Copyright 2008 Annual Reviews. (B) In the optical readout nanopore design
ach nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DNA
ragment through the nanopore. Reprinted from ref 82. Copyright 2010 American Chemical Society.
Nanopore DNA sequencing using electronic measurements and optical readout as detection methods.(A)In electronic nanopore schemes, signal is obtained through ionic current,73 tunneling
current, and voltage difference measurements. Each method must produce a characteristic signal to differentiate the four DNA bases. (B) In the optical readout nanopore design, each nucleotide
is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DNA fragment through the nanopore.
Single Molecule III: Oxford Nanopores
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics
Oxford Nanopores MinIon
“It’s kind of a cute device,” says Jaffe of the MinION, which is roughly the size and shape of a packet of chewing
gum. “It has pretty lights and a fan that hums pleasantly, and plugs into a USB drive.” But his technical review is
mixed. From http://www.nature.com/news/data-from-pocket-sized-genome-sequencer-unveiled-1.14724
Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics

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Evolution of DNA Sequencing - talk by Jonathan Eisen for the Bodega Workshop in Applied Phylogenetics

  • 1. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics ! Evolution of Sequencing ! Workshop in Applied Phylogenetics March 9, 2014 Bodega Bay Marine Lab ! Jonathan A. Eisen UC Davis Genome Center
  • 2. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Review Papers Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303. doi: 10.1146/annurev- anchem-062012-092628.
  • 3. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Review Papers Next-Generation DNA Sequencing Methods Elaine R. Mardis Departments of Genetics and Molecular Microbiology and Genome Sequencing Center, Washington University School of Medicine, St. Louis MO 63108; email: emardis@wustl.edu k links to ontent online, his volume articles ve search ther Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402 First published online as a Review in Advance on June 24, 2008 The Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org Annu.Rev.Genom.HumanGenet.2008.9: byUniversidadNacionalAutonom
  • 4. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Open Access Papers of Interest • http://www.microbialinformaticsj.com/content/2/1/3/ • http://www.hindawi.com/journals/bmri/2012/251364/abs/ • http://m.cancerpreventionresearch.aacrjournals.org/ content/5/7/887.full
  • 5. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Approaching to NGS Discovery of DNA structure (Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31) 1953 Sanger sequencing method by F. Sanger (PNAS ,1977, 74: 560-564) 1977 PCR by K. Mullis (Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73) 1983 Development of pyrosequencing (Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365) 1993 1980 1990 2000 2010 Single molecule emulsion PCR 1998 Human Genome Project (Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351) Founded 454 Life Science 2000 454 GS20 sequencer (First NGS sequencer) 2005 Founded Solexa 1998 Solexa Genome Analyzer (First short-read NGS sequencer) 2006 GS FLX sequencer (NGS with 400-500 bp read lenght) 2008 Hi-Seq2000 (200Gbp per Flow Cell) 2010 Illumina acquires Solexa (Illumina enters the NGS business) 2006 ABI SOLiD (Short-read sequencer based upon ligation) 2007 Roche acquires 454 Life Sciences (Roche enters the NGS business) 2007 NGS Human Genome sequencing (First Human Genome sequencing based upon NGS technology) 2008 Miseq Roche Jr Ion Torrent PacBio Oxford From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing Sequencing Technology Timeline
  • 6. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation I: Manual Sequencing
  • 7. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Maxam-Gilbert Sequencing http://www.pnas.org/content/74/2/560.full.pdf
  • 8. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sanger Sequencing of PhiX174 http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC431765/
  • 9. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sanger Sequencing
  • 10. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sanger Sequencing
  • 11. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Nobel Prize 1980: Berg, Gilbert, Sanger
  • 12. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation II: Automated Sanger
  • 13. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Automation of Sanger Part I Sanger method with labeled dNTPs The Sanger mehtods is based on the idea that inhibitors can terminate elongation of DNA at specific points
  • 14. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Automation of Sanger Part II
  • 15. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Automated Sanger Highlights • 1991: ESTs by Venter • 1995: Haemophilus influenzae genome • 1996: Yeast, archaeal genomes • 1999: Drosophila genome • 2000: Arabidopsis genome • 2000: Human genome • 2004: Shotgun metagenomics
  • 16. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation III: Clusters not Clones
  • 17. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation III = “NextGen”
  • 18. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen Sequencing OutlineNext-generation sequencing platforms Isolation and purification of target DNA Sample preparation Library validation Cluster generation on solid-phase Emulsion PCR Sequencing by synthesis with  3’-blocked reversible terminators Pyrosequencing Sequencing by ligation Single colour imaging Sequencing by synthesis with  3’-unblocked reversible terminators AmplificationSequencingImaging Four colour imaging Data analysis Roche 454Illumina GAII ABi SOLiD Helicos HeliScope From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/ high-throughput-equencing
  • 19. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing NextGen #1: 454
  • 20. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII NextGen #1: Roche 454 From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing
  • 21. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII NextGen #1: Roche 454 From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing
  • 22. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Roche 454 Wokflow From http://acb.qfab.org/acb/ws09/presentations/Day1_DMiller.pdf http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  • 23. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics a b DNA library preparation Emulsion PCR A A A B B B 4.5 hours 8 hours Ligation Selection (isolate AB fragments only) •Genome fragmented by nebulization •No cloning; no colony picking •sstDNA library created with adaptors •A/B fragments selected using avidin-biotin purification gDNA sstDNA library gDNA fragmented by nebulization or sonication Fragments are end- repaired and ligated to adaptors containing universal priming sites Fragments are denatured and AB ssDNA are selected by avidin/biotin purification (ssDNA library) From Mardis 2008. Annual Rev. Genetics 9: 387. Roche 454 Step 1: Libraries
  • 24. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Anneal sstDNA to an excess of DNA capture beads Emulsify beads and PCR reagents in water-in-oil microreactors Clonal amplification occurs inside microreactors Break microreactors and enrich for DNA-positive beads b c Emulsion PCR Sequencing A A B B 8 hours 7.5 hours only) with adaptors •A/B fragments selected using avidin-biotin purification gDNA sstDNA library sstDNA library Bead-amplified sstDNA library From Mardis 2008. Annual Rev. Genetics 9: 387. Roche 454 Step 2: Emulsion PCR
  • 25. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Anneal sstDNA to an excess of DNA capture beads Emulsify beads and PCR reagents in water-in-oil microreactors Clonal amplification occurs inside microreactors Break microreactors and enrich for DNA-positive beads Amplified sstDNA library beads Quality filtered bases c Sequencing 7.5 hours sstDNA library Bead-amplified sstDNA library •Well diameter: average of 44 µm •400,000 reads obtained in parallel •A single cloned amplified sstDNA bead is deposited per well 390 Mardis From Mardis 2008. Annual Rev. Genetics 9: 387. Roche 454 Step 3: Pyrosequencing
  • 26. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing 44 µm Pyrosequecning Reads are recorded as flowgrams Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Nature Reviews genetics, 2010, 11: 31-46 Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/ cosentia/high-throughput- equencing Roche 454 Step 3: Pyrosequencing
  • 27. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Roche 454 Key Issues • Number of repeated nucleotides estimated by amount of light ... many errors • Reasonable number of failures in EM- PCR and other steps
  • 28. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Roche 454 Evolution http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  • 29. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen #2: Solexa Sequecning by synthesis with reversible terminator From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput- equencing
  • 30. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sequecning by synthesis with reversible terminator From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing NextGen #2: Solexa Illumina
  • 31. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen #2: Illumina Accessories Cluster station Genome Analyzer IIxPaired-end module Linux server Bioanalyzer 2100 Instrumentation le tion ers ation ng by esis sis ne ction GAII h hput From Slideshare presentation of Cosentino Cristian http:// www.slideshare. net/cosentia/ high-throughput- equencing
  • 32. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Outline Clusters amplification Clusterstation Wash cluster station Clustergeneration Linearization, Blocking and primer Hybridization Read 1 Prepare read 2 Read 2 GAIIx&PE SBSsequencing Pipeline base call Data analysis Sample preparation and library validation Analysis Sequencing workflow Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing
  • 33. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Step 1: Prep & Attach DNA continues for a specific number of cycles, as de- termined by user-defined instrument settings, which permits discrete read lengths of 25–35 read and a quality checking pipeline evaluates the Illumina data from each run, removing poor-quality sequences. Adapter DNA fragment Dense lawn of primers Adapter Attached DNA Adapters Prepare genomic DNA sample Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. Attach DNA to surface Bind single-stranded fragments randomly to the inside surface of the flow cell channels. Nucleotides a From Mardis 2008. Annual Rev. Genetics 9: 387. Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR
  • 34. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Step 2: Clusters by Bridge PCR Attached Prepare genomic DNA sample Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. Attach DNA to surface Bind single-stranded fragments randomly to the inside surface of the flow cell channels. Bridge amplification Add unlabeled nucleotides and enzyme to initiate solid- phase bridge amplification. Denature the double stranded molecules Nucleotides Figure 2 The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and all four fluorescently labeled, 3′-OH blocked nucleotides are added to the flow cell with DNA polymerase. The cluster strands are extended by one nucleotide. Following the incorporation step, the unused nucleotides and DNA polymerase molecules are washed away, a scan buffer is added to the flow cell, and the optics system scans each lane of the flow cell by imaging units called tiles. Once imaging is completed, chemicals that effect cleavage of the fluorescent labels and the 3′-OH blocking groups are added to the flow cell, which prepares the cluster strands for another round of fluorescent nucleotide incorporation. 392 Mardis From Mardis 2008. Annual Rev. Genetics 9: 387. From : http://seqanswers.com/forums/showthread.php?t=21. Steps 2-6: Cluster Generation by Bridge Amplification. In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illumina's "cluster station", an automated flow cell processor.
  • 35. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Clusters
  • 36. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics tween 2–4 Gb of DNA sequence data. Once different from that already established for b Laser First chemistry cycle: determine first base To initiate the first sequencing cycle, add all four labeled reversible terminators, primers, and DNA polymerase enzyme to the flow cell. Image of first chemistry cycle After laser excitation, capture the image of emitted fluorescence from each cluster on the flow cell. Record the identity of the first base for each cluster. Sequence read over multiple chemistry cycles Repeat cycles of sequencing to determine the sequence of bases in a given fragment a single base at a time. Before initiating the next chemistry cycle The blocked 3' terminus and the fluorophore from each incorporated base are removed. GCTGA... From Mardis 2008. Annual Rev. Genetics 9: 387. Illumina Step 3: Sequencing by Synthesis From : http://seqanswers.com/forums/showthread.php?t=21. Steps 7-12: Sequencing by Synthesis. A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases.
  • 37. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics SBS technology Sample reparation Clusters mplification quencing by synthesis Analysis pipeline troduction umina GAII High hroughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing Illumina Step 3: Sequencing by Synthesis
  • 38. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Laser DNA polymerase enzyme to the flow cell. Image of first chemistry cycle After laser excitation, capture the image of emitted fluorescence from each cluster on the flow cell. Record the identity of the first base for each cluster. Sequence read over multiple chemistry cycles Repeat cycles of sequencing to determine the sequence of bases in a given fragment a single base at a time. Before initiating next chemistry cy The blocked 3' termi and the fluorophore from each incorpora base are removed. GCTGA... Figure 2 (Continued ) www.annualreviews.org • Next-Generation DNA Sequencing Methods 393 From Mardis 2008. Annual Rev. Genetics 9: 387. Illumina Step 3: Cycling
  • 39. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Evolution http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  • 40. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics MiSeq Dx
  • 41. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics HiSeq x Ten
  • 42. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics HiSeq x Ten
  • 43. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen #3: 454: ABI SolidSequecning by ligation Sanger method Roche 454 - HeliScope Nanopore ABi SOLiD Illumina GAII From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing
  • 44. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics ABI Solid Details G09-20 ARI 25 July 2008 14:57 A C G T 1stbase 2nd base A C G T 3'TAnnnzzz5' 3'TCnnnzzz5' 3'TGnnnzzz5' 3'TTnnnzzz5' Cleavage site Di base probesSOLiD™ substrate 3' TA AT Universal seq primer (n) 3' P1 adapter Template sequence POH Universal seq primer (n–1) Ligase Phosphatase + 1. Prime and ligate 2. Image 4. Cleave off fluor 5. Repeat steps 1–4 to extend sequence 3' Universal seq primer (n–1) 1. Melt off extended sequence 2. Primer reset3' AA AC G G GG C C C T AA A GG CC T TTT 6. Primer reset 7. Repeat steps 1–5 with new primer 8. Repeat Reset with , n–2, n–3, n–4 primers TA AT AT 3' TA AT 3' Excite Fluorescence Cleavage agent P HO TA AA AG AC AAAT TT TC TG TT AC TG CG GC 3' 3. Cap unextended strands 3' PO4 1 2 3 4 5 6 7 ... (n cycles)Ligation cycle 3' 3'1 μm bead 1 μm bead 1 μm bead –1 Universal seq primer (n)1 Primer round 1 Template Primer round 2 1 base shift Glass slide 3'5' Template sequence 1 μm bead P1 adapter Read position 35343332313029282726252423222120191817161514131211109876543210 a The ligase-mediated sequencing approach of the Applied Biosystems SOLiD sequencer. In a manner similar to Roche/454 emulsion PCR amplification, DNA fragments for SOLiD sequencing are amplified on the surfaces of 1-μm magnetic beads to provide sufficient signal during the sequencing reactions, and are then deposited onto a flow cell slide. Ligase-mediated sequencing begins by annealing a primer to the shared adapter sequences on each amplified fragment, and then DNA ligase is provided along with specific fluorescent- labeled 8mers, whose 4th and 5th bases are encoded by the attached fluorescent group. Each ligation step is followed by fluorescence detection, after which a regeneration step removes bases from the ligated 8mer (including the fluorescent group) and concomitantly prepares the extended primer for another round of ligation. (b) Principles of two- base encoding. Because each fluorescent group on a ligated 8mer identifies a two-base combination, the resulting sequence reads can be screened for base-calling errors versus true polymorphisms versus single base deletions by aligning the individual reads to a known high- quality reference sequence. From Mardis 2008. Annual Rev. Genetics 9: 387.
  • 45. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics ABI Solid Evolution http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  • 46. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Complete Genomics REVIEW ased the number of false positive gene ely reduced the number gene candidates sitosterolemia phenotype were determined after comparison of the patient’s genome to a collection of reference genomes. omplete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DNB f the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a set onding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. Reprinted with pyright XXXX American Association for the Advancement of Science. gure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DN nthesis, and dimensions of the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a Figure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DNB synthesis, and dimensions of the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a set of common probes corresponding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
  • 47. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Comparison in 2008 Roche (454) Illumina SOLiD Chemistry Pyrosequencing Polymerase-based Ligation-based Amplification Emulsion PCR Bridge Amp Emulsion PCR Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb Mb/run 100 Mb 1300 Mb 3000 Mb Time/run 7 h 4 days 5 days Read length 250 bp 32-40 bp 35 bp Cost per run (total) $8439 $8950 $17447 Cost per Mb $84.39 $5.97 $5.81 From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/ CSHL_nextgen.ppt
  • 48. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Comparison in 2012 Roche (454) Illumina SOLiD Chemistry Pyrosequencing Polymerase-based Ligation-based Amplification Emulsion PCR Bridge Amp Emulsion PCR Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb Mb/run 100 Mb 1300 Mb 3000 Mb Time/run 7 h 4 days 5 days Read length 250 bp 32-40 bp 35 bp Cost per run (total) $8439 $8950 $17447 Cost per Mb $84.39 $5.97 $5.81 From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/ CSHL_nextgen.ppt
  • 49. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Bells and Whistles
  • 50. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Multiplexing From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn
  • 51. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Multiplexing http://res.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf
  • 52. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Small Amounts of DNA http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4
  • 53. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Capture MethodsHigh throughput sample preparation Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput Nature Methods, 2010, 7: 111-118 RainDance Microdroplet PCR Roche Nimblegen Salid-phase capture with custom- designed oligonucleotide microarray Reported 84% of capture efficiency Reported 65-90% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing
  • 54. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics High throughput sample preparation Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput Agilent SureSelect Solution-phase capture with streptavidin-coated magnetic beads Reported 60-80% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/ high-throughput-equencing Capture Methods
  • 55. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Paired Ends Paired-end technology Paired-end sequencing works into GA and uses chemicals from the PE module to perform cluster amplification of the reverse strandSample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing
  • 56. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Moleculo Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes Sequence w/ Illumina Assemble seqs w/ same codes
  • 57. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation III+: Faster w/ Clusters
  • 58. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Ion Torrent PGM
  • 59. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Applied Biosystems Ion Torrent PGM
  • 60. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Applied Biosystems Ion Torrent PGM Workflow similar to that for Roche/454 systems. ! Not surprising, since invented by people from 454.
  • 61. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Ion Torrent pH Based Sequencing Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.
  • 62. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Ion Torrent Evolution
  • 63. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation IV: Single Molecule
  • 64. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Single Molecule I: Helicos 3rd Generation Sequencing by low for lecular scent_sequencing
  • 65. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Single Molecule II: Pacific Biosciences
  • 66. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Single Molecule II: Pacific Biosciences Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.
  • 67. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Analytical Chemistry REVIEW Φ29 polymerase. Each amplified product of a circularized fragment is called a DNA nanoball (DNB). DNBs are selectively attached to a hexamethyldisilizane (HMDS) coated silicon chip that is photolithographically patterned with aminosilane active sites. Figure 3A illustrates the DNB array design. The use of the DNBs coupled with the highly patterned array offers several advantages. The production of DNBs increases signal intensity by simply increasing the number of hybridization sites available for probing. Also, the size of the DNB is on the same length scale as the active site or “sticky” spot patterned on Each hybridization and ligation cycle is followed by fluorescent imaging of the DNB spotted chip and subsequently regeneration of the DNBs with a formamide solution. This cycle is repeated until the entire combinatorial library of probes and anchors is examined. This formula of the use of unchained reads and regeneration of the sequencing fragment reduces reagent con- sumption and eliminates potential accumulation errors that can arise in other sequencing technologies that require close to completion of each sequencing reaction.19,52,53 Complete Genomics showcased their DNB array and cPAL Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at the bottom surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing template. The corresponding temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright 2009 American Association for the Advancement of Science. Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at the bottom surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing template. The corresponding temporal fluorescence detection with respect to each of the five incorporation steps is shown below. From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011. Single Molecule II: Pacific Biosciences
  • 68. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Why Finish Genomes? The Value of Finished Bacterial Genomes Why Are Finished Genomes So Important? When Sanger sequencing was the only available sequencing technique, it was expensive — but not unusual — to improve genome drafts until they were good enough to be considered finished. With the availability of short-read sequencing technologies, draft genomes became cheap and easy to produce, and the majority of researchers skipped the more labor- and time-intensive task of finishing genomes, with the realization that critical data may be missing (Figure 3). Finished genomes are crucial for understanding microbes and advancing the field of microbiology3 because: • Functional genomic studies demand a high-quality, complete genome sequence as a starting point • Comparative genomics is meaningful only in terms of complete genome sequences • Understanding genome organization provides biological insights • Microbial forensics requires at least one complete reference genome sequence • Finished genomes aid in microbial outbreak source identification and phylogenetic analysis • A complete genome is a permanent scientific resource Figure 3: History of drafted vs. finished genomes (adapted from ref. 2). Microbial Genetics Using SMRT Sequencing 0 2000 4000 6000 8000 10000 12000 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Numberofgenomes Drafted Bacterial Genomes Finished Bacterial Genomes
  • 69. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Why Finish Genomes JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 6403–6405 Vol. 184, No. 23 0021-9193/02/$04.00ϩ0 DOI: 10.1128/JB.184.23.6403–6405.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. DIALOG The Value of Complete Microbial Genome Sequencing (You Get What You Pay For) Claire M. Fraser,* Jonathan A. Eisen, Karen E. Nelson, Ian T. Paulsen, and Steven L. Salzberg The Institute for Genomic Research, Rockville, Maryland 20850 Since the publication of the complete Haemophilus influen- zae genome sequence in July 1995 (4), the field of microbiology has been one of the largest beneficiaries of the breakthroughs in genomics and computational biology that made this accom- plishment possible. When the 1.8-Mbp H. influenzae project began in 1994, it was not certain that the whole-genome shot- gun sequencing strategy would succeed because it had never been attempted on any piece of DNA larger than an average lambda clone (ϳ40 kbp) (9). During the past 7 years, progress in DNA sequencing tech- nology, the design of new vectors for library construction for use in shotgun sequencing projects, significant improvements in closure and finishing strategies, and more sophisticated and robust methods for gene finding and annotation have dramat- ically reduced the time required for each stage of a genome organisms to be sampled because of the cost savings that would come from not taking each project to completion. While this strategy does achieve a cost savings, today it is only approxi- mately 50%, and this comes at a cost in terms of the quality and utility of the finished product. A complete genome sequence represents a finished product in which the order and accuracy of every base pair have been verified. In contrast, a draft sequence, even one of high cov- erage, represents a collection of contigs of various sizes, with unknown order and orientation, that contain sequencing errors and possible misassemblies. As stated by Selkov et al. in a 2000 paper on a draft sequence of Thiobacillus ferrooxidans, “It is clear that such sequencing. . .produces more errors than com- plete genome sequencing. . . . The current error rate is esti- mated to be 1 per 1,000 to 2,000 base pairs vs. 1 in 10,000 base http://jb.asm.orDownloadedfrom http://jb.asm.org/content/184/23/6403.full
  • 70. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics HGAP Assembly from PacBio PacBio assembly CDC assembly Illumina assemblySanger validation HGAP Assembler for PacBio Data http://www.pacificbiosciences.com/pdf/ microbial_primer.pdf
  • 71. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Detecting Modified Bases Page 2 www.pacb.com/basemod processes. The potential benefits of detecting base modification, using SMRT sequencing, include: Single-base resolution detection of a wide the presence of a modified base in the DNA template 3 . This is observable as an increased space between fluorescence pulses, which is called the interpulse duration (IPD), as shown in Figure 2. Figure 2. Principle of detecting modified DNA bases during SMRT sequencing. The presence of the modified base in the DNA template (top), shown here for 6-mA, results in a delayed incorporation of the corresponding T nucleotide, i.e. longer interpulse duration (IPD), compared to a control DNA template lacking the modification (bottom). 3 http://www.pacificbiosciences.com/pdf/microbial_primer.pdf
  • 72. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. By measuring that current it is possible to identify the molecule in question. For example, this system can be used to distinguish the four standard DNA bases and G, A, T and C, and also modified bases. It can be used to identify target proteins, small molecules, or to gain rich molecular information for example to distinguish the enantiomers of ibuprofen or molecular binding dynamics. From Oxford Nanopores Web Site Single Molecule III: Oxford Nanopores
  • 73. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics • Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore. (A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall). tical Chemistry RE 6. Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of RHL protein nanopore mutant depicting the position extrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the loca inines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleav tides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein na ows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded th n nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Co Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall). Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore.(A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases ar identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall). Single Molecule III: Oxford Nanopores From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
  • 74. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Analytical Chemistry REVIEW would result, in theory, in detectably altered current flow through he pore. Theoretically, nanopores could also be designed to measure tunneling current across the pore as bases, each with a istinct tunneling potential, could be read. The nanopore ap- lipid bilayer, using ionic current blockage method. The author predicted that single nucleotides could be discriminated as lon as: (1) each nucleotide produces a unique signal signature; (2 the nanopore possesses proper aperture geometry to accommo igure 5. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A) In electronic nanopore schemes ignal is obtained through ionic current,73 tunneling current,78 and voltage difference79 measurements. Each method must produce a characteristic signa o differentiate the four DNA bases. Reprinted with permission from ref 83. Copyright 2008 Annual Reviews. (B) In the optical readout nanopore design ach nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DNA ragment through the nanopore. Reprinted from ref 82. Copyright 2010 American Chemical Society. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods.(A)In electronic nanopore schemes, signal is obtained through ionic current,73 tunneling current, and voltage difference measurements. Each method must produce a characteristic signal to differentiate the four DNA bases. (B) In the optical readout nanopore design, each nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DNA fragment through the nanopore. Single Molecule III: Oxford Nanopores From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
  • 75. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Oxford Nanopores MinIon “It’s kind of a cute device,” says Jaffe of the MinION, which is roughly the size and shape of a packet of chewing gum. “It has pretty lights and a fan that hums pleasantly, and plugs into a USB drive.” But his technical review is mixed. From http://www.nature.com/news/data-from-pocket-sized-genome-sequencer-unveiled-1.14724
  • 76. Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics