EVE161 Lecture 2

EVE 161: 
Microbial Phylogenomics
!

Lecture #2:
Evolution of Sequencing
!
UC Davis, Winter 2014
Instructor: Jonathan Eisen

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

!1

Where we are going and where we have been

• Previous lecture:
! 1. Introduction
• Current Lecture:
! 2. Sequencing Evolution
• Next Lecture:
! 3. Era I: The Tree of Life


!2

Main Paper


!3

Annu. Rev. Genom. Human Genet. 2008.9:
by Universidad Nacional Autonom

ther

k links to
ontent online,

his volume

articles
ve search

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

Annu. Rev. Genomics Hum. Genet. 2008.
9:387–402
First published online as a Review in Advance on
June 24, 2008
The for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
Annual Review of Genomics and Human Genetics
Slides
Slides for UC at EVE161 Course Taught by Jonathan Eisen
is online Davisgenom.annualreviews.orgWinter 2014

!4

Many other papers of interest
• http://www.microbialinformaticsj.com/
content/2/1/3/
• http://www.hindawi.com/journals/bmri/
2012/251364/abs/
• http://
m.cancerpreventionresearch.aacrjourna
ls.org/content/5/7/887.full


!5

Sequencing Technology


!6

Key Issues
•
•
•
•
•
•

Cost / bp
Read length
Paired end
Ease of feeding
Error profiles
Barcoding potential


!7

Timeline


!8

Timeline

1977

2010

Sanger sequencing method by F. Sanger
(PNAS ,1977, 74: 560-564)

1983
1953

2000

1990

1980

Approaching to NGS

PCR by K. Mullis
(Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73)

Discovery of DNA structure

Human Genome Project

(Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31)

(Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351)

1993

Development of pyrosequencing
(Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365)

Single molecule emulsion PCR

1998

Founded Solexa

1998

Founded 454 Life Science

2000

454 GS20 sequencer
(First NGS sequencer)

2005

Solexa Genome Analyzer
(First short-read NGS sequencer)

Illumina acquires Solexa
(Illumina enters the NGS business)

2006
2006

ABI SOLiD
(Short-read sequencer based upon ligation)

Roche acquires 454 Life Sciences

From Slideshare presentation of Cosentino Cristian
http://www.slideshare.net/cosentia/highthroughput-equencing

(Roche enters the NGS business)

2007
2007

GS FLX sequencer
(NGS with 400-500 bp read lenght)

NGS Human Genome sequencing
(First Human Genome sequencing based upon NGS technology)

2008
2008

Hi-Seq2000
(200Gbp per Flow Cell)


2010

!9

Timeline
1977

2010

Sanger sequencing method by F. Sanger
(PNAS ,1977, 74: 560-564)

1983
1953

2000

1990

1980

Approaching to NGS

PCR by K. Mullis
(Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73)

Discovery of DNA structure
(Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31)

Human Genome Project
(Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351)

1993

Development of pyrosequencing
(Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365)

Single molecule emulsion PCR

1998

Founded Solexa

1998

Founded 454 Life Science

2000

454 GS20 sequencer
(First NGS sequencer)

2005

Solexa Genome Analyzer
(First short-read NGS sequencer)

Illumina acquires Solexa
(Illumina enters the NGS business)

2006
2006

ABI SOLiD
(Short-read sequencer based upon ligation)

Roche acquires 454 Life Sciences
(Roche enters the NGS business)

2007
2007

GS FLX sequencer
(NGS with 400-500 bp read lenght)

NGS Human Genome sequencing
(First Human Genome sequencing based upon NGS technology)

http://www.slideshare.net/cosentia/highthroughput-equencing

2008
2008

Hi-Seq2000
(200Gbp per Flow Cell)

2010


Miseq
Roche Jr
Ion Torrent
PacBio
Oxford

!10

Generation I: Manual


!11

Maxam-Gilbert Sequencing


!12


http://www.pnas.org/content/74/2/560.full.pdf


!13



!14



!15

Sanger Sequencing

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/


!16

PhiX174


!17

Sanger Sequencing


!18

Sanger Sequencing


!19

Nobel Prize 1980


!20

Generation II: Automation


!21

Sanger method with labeled dNTPs

Automation I

The Sanger mehtods is based on the idea that inhibitors can
terminate elongation of DNA at specific points


!22

Sanger method with labeled dNTPs

Automation I

The Sanger mehtods is based on the idea that inhibitors can
terminate elongation of DNA at specific points


!23

Automation 2


!24

Automated Sanger Highlights
•
•
•
•
•
•
•

1991: ESTs by Venter
1995: Haemophilus influenzae genome
1996: Yeast, archaea genomes
1999: Drosophila genome
2000: Arabidopsis genome
2000: Human genome
2004: Shotgun metagenomics


!25

Generation III: Clusters not clones


!26

Generation III: Clusters not clones


!27

Next Gen Sequencingplatforms
Outline
Next-generation sequencing
From Slideshare presentation of
Cosentino Cristian
http://www.slideshare.net/cosentia/
high-throughput-equencing

Isolation and purification of
target DNA

Sample preparation

Amplification

Emulsion PCR

Sequencing by synthesis
with 3’-blocked reversible
terminators

Pyrosequencing

Imaging

Cluster generation
on solid-phase

Sequencing

Library validation

Sequencing by ligation

Four colour imaging

with 3’-unblocked reversible
terminators

Single colour imaging

Data analysis

Illumina GAII

Roche 454

ABi SOLiD


Helicos HeliScope

!28

454


!29

454

Roche 454
Sanger
method
ABi SOLiD
Illumina GAII

Pyrosequencing
From Slideshare
presentation of Cosentino
Cristian
http://www.slideshare.net/
cosentia/high-throughputequencing

HeliScope
Nanopore


!30

Roche 454

Roche 454
Sanger
method
ABi SOLiD
Illumina GAII

Pyrosequencing

From Slideshare
Cristian

HeliScope
Nanopore


!31

454 -> Roche
• 1st “Next-generation” sequencing
system to become commercially
available, in 2004
• Uses pyrosequencing
• Polymerase incorporates nucleotide
• Pyrophosphate released
• Eventually light from luciferase released

• Three main steps in 454 method
• Library prep
• Emulsion PCR
• Sequencing

!32

454


!33

454 Step1: Library Prep
a
DNA library preparation
4.5 hours
Ligation

B

•Genome fragmented
by nebulization

A

•No cloning; no colony
picking

Selection
(isolate AB
fragments
only)
A

B

A

B

•sstDNA library created
with adaptors
•A/B fragments selected
using avidin-biotin
purification

gDNA
gDNA
b fragmented by
nebulization or
Emulsion PCR
sonication
8 hours

sstDNA library
Fragments are endrepaired 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.

!34

only)
A

B

with adaptors
•A/B fragments selected
using avidin-biotin
purification

454 Step 2: Emulsion PCR
A

B

gDNA

sstDNA library

b
Emulsion PCR
8 hours

Anneal sstDNA to an excess of
DNA capture beads

sstDNA library

c

Emulsify beads and PCR
reagents in water-in-oil
microreactors

Clonal amplification occurs
inside microreactors

Break microreactors and
enrich for DNA-positive
beads

Bead-amplified sstDNA library


Sequencing
7.5 hours


!35

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

454 Step 3: Sequencing

sstDNA library

Bead-amplified sstDNA library

c
Sequencing
7.5 hours

•Well diameter: average of 44 µm
•400,000 reads obtained in parallel
•A single cloned amplified sstDNA
bead is deposited per well

Amplified sstDNA library beads

390

Mardis

Quality filtered bases


!36

454 Step 3: Sequencing
Pyrosequencing

Roche 454
Sanger
method

Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402
Nature Reviews genetics, 2010, 11: 31-46

-

44 µm

ABi SOLiD
Illumina GAII
HeliScope
Nanopore

From Slideshare
Cristian

Pyrosequecning

Reads are recorded as flowgrams


!37

454 Key Issues
• Number of repeated nucleotides
estimated by amount of light ... many
errors
• Reasonable number of failures in EMPCR and other steps
• Systems
•
•
•
•
•

GS20
FLX
FLX Titanium
FLX Titanium XL
Junior

!38


!39

X

DISCONTINUED



!40

Solexa


!41

Sequecning by synthesis with reversible terminator

Solexa

From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/
cosentia/highthroughput-equencing


!42

Sequecning by synthesis with reversible terminator

Illumina

From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/


!43

Illumina
•
•
•
•

1st system released in 2006 by Solexa
Acquired by Illumina in 2007
Systems
•
•
•
•
•

GA
GAII
HiSeqs
HiScan
MiSeq

!44

Instrumentation

GAII

Accessories

ction

le
tion

Bioanalyzer 2100

Cluster station

ers
ation

ng by
esis

sis
ne

h
hput

Paired-end module

Genome Analyzer IIx

Linux server


!45

Illumina


!46

Illumina
a

b

Illumina’s library-preparation work flow
DNA fragments

OH OH
Blunting by fill-in
and exonuclease

P
P
P

P
A
A

Phosphorylation

P7 P5
Grafted
flow ce

P
Addition of
A-overhang
Ligation
to adapters

P

First
denatura

c

3' 5'

O

Fluor

!47

mina GAII

oduction

Flow cell

Illumina Flow Cell

ample
paration

usters
lification

encing by
nthesis

nalysis
peline

High
oughput

From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/
cosentia/highthroughput-equencing !48

13 May 2013

16:4

Illumina
b

library-preparation work flow
DNA fragments

OH OH
Blunting by fill-in
and exonuclease

P
A
A

Phosphorylation

First cycle
annealing

First cycle
extension

Second cycle
denaturation

Fluor

HN
O N
O
PPP
3'

n = 35
total

Cleavage
site

Block
Second cycle
denaturation

Incorporate
Detect
Deblock
Cleave fluor
O
HN
5'
DNA O N
O O
3'

5'

Denaturation

P

O

Excitation

Initial
extension

P

5'

Emission

Template
hybridization

Addition of
A-overhang
Ligation
to adapters

3'

P7 P5
Grafted
flow cell

First
denaturation

P

Second cycle
annealing

Second cycle
extension

Cluster
amplification

P5
linearization

Block with
ddNTPs

x

OH free 3' end

Denaturation and
sequencing primer
hybridization


brary-construction process. (b) Illumina cluster generation by bridge ampliﬁcation. (c) Sequencing by synthesis with

!49

continues for a specific number of cycles, as determined 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.

Illumina Step 1: Attach DNA
a

Adapter
DNA fragment

DNA

Dense lawn
of primers
Adapter

Adapters

Prepare genomic DNA sample

Attach DNA to surface

Randomly fragment genomic DNA
and ligate adapters to both ends of
the fragments.

Bind single-stranded fragments
randomly to the inside surface
of the flow cell channels.

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
Nucleotides
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
Attached
!50

Prepare genomic DNA sample

Attach DNA to surface

Randomly fragment genomic DNA
and ligate adapters to both ends of
the fragments.

Bind single-stranded fragments
randomly to the inside surface
of the flow cell channels.

Illumina Step 2: Bridge PCR
Nucleotides

Attached

Bridge amplification
Add unlabeled nucleotides
and enzyme to initiate solidphase bridge amplification.

Denature the double
stranded molecules

The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and
Figure 2
′
all four Illumina sequencing-by-synthesis approach. Cluster strands created added toamplification are with DNA all four fluorescently
The fluorescently labeled, 3 -OH blocked nucleotides are by bridge the flow cell primed and polymerase. The
cluster strands are extended by are added to the flowFollowing the incorporationcluster strands are extended by one and
labeled, 3′ -OH blocked nucleotides one nucleotide. cell with DNA polymerase. The step, the unused nucleotides
nucleotide. Following the incorporation step, the unused scan buffer DNA polymerase flow cell, and the optics system
DNA polymerase molecules are washed away, a nucleotides andis added to the molecules are washed away, a scan buffer is
added to the flow cell, and the cell system scans units called flow Once imaging is completed, chemicals that effect
scans each lane of the flow opticsby imaging each lane of thetiles.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
′
cleavagestrands for another round of fluorescent nucleotideblocking groups are added to the flow cell, which prepares the
cluster of the fluorescent labels and the 3 -OH incorporation.
cluster strands for another round of fluorescent nucleotide incorporation
!51
392

Mardis


Clusters


!52

sity of California - Davis on 01/09/14. For personal use only.

P

Ligation
to adapters

Sequencing by Synthesis
First
denaturation
3'

c

5'

HN
O N
O
PPP
Emission

A
C
T

G
Excitation

C
A

Second cycle
denaturation

Sec
an

Cluster
amplification

line

Fluor

O

3'

Fi
an

Cleavage
site

Block
Incorporate
Detect
Deblock
Cleave fluor

T
O

G
A

HN
5'
DNA O N
O O

T
G
C

3'
5'

x

OH free 3' end


Figure 3


!53

lengths for SOLiD are user deﬁned between
25–35 bp, and each sequencing run yields between 2–4 Gb of DNA sequence data. Once

from a capillary sequencer and (b) each nextgeneration read type has a unique error model
different from that already established for

Illumina Step 3: Sequencing

b
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

Before initiating the
next 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.

The blocked 3' terminus
and the fluorophore
from each incorporated
base are removed.

Laser

GCTGA...
Slides for read over multiple chemistry by Jonathan Eisen Winter 2014
Sequence UC Davis EVE161 Course Taughtcycles

!54

umina GAII

Illumina
SBS technologySBS

troduction

Sample
reparation

Clusters
mplification

quencing by
synthesis

Analysis
pipeline

High
hroughput

From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/


!55

Illumina


!56

ABI Solid


!57

ABI Solid
Sequecning by ligation

ABi SOLiD
Sanger
method
Roche 454
Illumina GAII
HeliScope
Nanopore

From Slideshare
presentation of
Cosentino Cristian
http://
www.slideshare.net/


!58

G09-20

ARI

25 July 2008

14:57

ABI Solid Details
The ligase-mediated sequencing approach of
the Applied Biosystems SOLiD sequencer. In a
SOLiD™ substrate
Di base probes
Template
manner similar to Roche/454 emulsion PCR
2nd base
3
'TTnnnzzz5
'
1 μm
amplification, DNA fragments for SOLiD
A C G T
bead 5'
Template sequence
3'
P1 adapter
A
3
'TGnnnzzz5
'
sequencing are amplified on the surfaces of 1C
G
μm magnetic beads to provide sufficient signal
3
'TCnnnzzz5
'
T
during the sequencing reactions, and are then
3
'TAnnnzzz5
'
deposited onto a flow cell slide. LigaseGlass slide
Cleavage site
mediated sequencing begins by annealing a
5. Repeat steps 1–4 to extend sequence
1. Prime and ligate
P OH
primer to the shared adapter sequences on
Ligation cycle 1
2
3
4
5
6
7 ... (n cycles)
+ Ligase
Primer round 1
each amplified fragment, and then DNA ligase
GC
TT
CT
GT
TT
CA
AT
TA
CG
GT
GA
CA
AA
AA
3' is provided along with specific fluorescentUniversal seq primer (n)
AT
3'
1 μm
3'
labeled 8mers, whose 4th and 5th bases are
bead
P1 adapter
Template sequence
TA
6. Primer reset
encoded by the attached fluorescent group.
2. Image
Excite
Fluorescence
Each ligation step is followed by fluorescence
Universal seq primer (n–1)
detection, after which a regeneration step
1. Melt off extended
3'
2. Primer reset
sequence
removes bases from the ligated 8mer
3'
1 μm
TA
3'
bead
(including the fluorescent group) and
3. Cap unextended strands
concomitantly prepares the extended primer
7. Repeat steps 1–5 with new primer
Phosphatase
for another round of ligation. (b) Principles of
PO
two-base encoding. Because each fluorescent
Primer round 2
1 base shift
3'
–1
group on a ligated 8mer identifies a two-base
Universal seq primer (n–1) A A C A C G T C A A T A C C
4. Cleave off fluor
3'
combination, the resulting sequence reads can
1 μm
3'
Cleavage agent
bead
T GT G C A G T T A T GG
be screened for base-calling errors versus true
HO
AT
polymorphisms versus single base deletions
P
3'
TA
by aligning the individual reads to a known
!59
8. Repeat Reset with , n–2, n–3, n–4 primers
From Mardis 2008. Annual Rev. Genetics 9: 387. high-quality reference sequence.
a

1st base

4

Read position


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

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

7h

4 days

5 days

Read length

250 bp

32-40 bp

35 bp

Cost per run
(total)
Cost per Mb

$8439

$8950

$17447

$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

!60

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

7h

4 days

5 days

Read length

250 bp

32-40 bp

35 bp

Cost per run
(total)
Cost per Mb

$8439

$8950

$17447

$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

!61

Bells and Whistles
•
•
•
•

Multiplexing
Paired end
Mate pair
????


!62

Multiplexing


!63

Small amounts of DNA


!64

Illumina GAII

Capture Methods

High throughput sample preparation

Introduction

Sample
preparation

RainDance
Microdroplet PCR

Roche Nimblegen
Salid-phase capture with customdesigned oligonucleotide microarray

Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
High
throughput

Reported 84% of
capture efficiency

http://www.slideshare.net/cosentia/high-throughput-equencing
Nature Methods, 2010, 7: 111-118

Reported 65-90% of capture efficiency

!65

Illumina GAII

Introduction

Sample
preparation

Capture preparation
Methods
High throughput sample
Agilent SureSelect
Solution-phase capture with
streptavidin-coated magnetic beads

Clusters
amplification
Sequencing by
synthesis
Analysis
pipeline
High
throughput

Reported 60-80% of capture efficiency

From Slideshare presentation of
Cosentino Cristian
http://www.slideshare.net/cosentia/
high-throughput-equencing


!66

Moleculo
DNA
Large fragments
Isolate and amplify

Sublibrary w/ unique barcodes
CACC

GGAA

TCTC

ACGT

AAGG

GATC

AAAA

Sequence w/ Illumina

Assemble seqs w/ same codes

!67

Generation 3.5
• Even faster


!68

Ion Torrent PGM


!69

Applied Biosystems Ion Torrent PGM
Workflow similar to
that for Roche/454
systems.
!
Not surprising,
since invented by
people from 454.


!70

Ion Torrent PGM


!71

Generation IV??
• Single molecule sequencing


!72

Pacific Biosciences


!73

Pacific Biosciences


!74


!75

Analytical Chemistry

Pacific Biosciences

REVIEW

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 a
Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of
temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright
single ZMW nanostructure containing a single DNA polymerase (Φ29) bound to the bottom
2009 American Association for the Advancement of Science.

glass surface. The ZMW and the confocal imaging system allow fluorescence detection only
Φ29 polymerase. Each amplified product of a circularized
Each hybridization and ligation cycle is followed by fluorescent
at theisbottom surface of each ZMW. (B) Representation of fluorescently subsequently regeneration
labeled nucleotide
fragment called a DNA nanoball (DNB). DNBs are selectively
imaging of the DNB spotted chip and
attached to a hexamethyldisilizane (HMDS) coated silicon chip template. The corresponding temporal is repeated
of the DNBs with a formamide solution. This cycle
substrate incorporation on to a sequencing
that is photolithographically patterned with aminosilane active
until the
library of probes and
fluorescence detection with respect to each of the fiveentire combinatorialthesteps is shown anchors is
below.
sites. Figure 3A illustrates the DNB array design.
examined. incorporation use of unchained reads and
This formula of

The use of the DNBs coupled with the highly patterned array
regeneration of the sequencing fragment reduces reagent conoffers several advantages. The production of DNBs et al. Analytical Chemistry 83: 4327. 2011. errors that can
sumption and eliminates potential accumulation
From Niedringhaus increases
signal intensity by simply increasing the number of hybridization
arise in other sequencing technologies that require close to
sites available for probing. Also, the for UC Davis EVE161 Course Taught by Jonathan Eisen Winter reaction.19,52,53
size of the DNB is on the
completion of each sequencing 2014
Slides
!76
Complete Genomics Winter 2014
same length scale as the active site orfor UC Davis EVE161 Course Taught by Jonathan Eisen showcased their DNB array and cPAL
“sticky” spot patterned on
Slides

Complete Genomics


!77

Complete Genomics
REVIEW

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.

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.

From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.

gure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent8
!7 DN
ased the number of false positive gene
sitosterolemia phenotype localize DNBs
after comparison of
nthesis, and dimensions of the patterned nanoarray used towere determinedTaught by the DNB array formation. (B) Illustration of sequencing with a
Slides for UC Davis EVE161 Course illustrate Jonathan Eisen Winter 2014

ely reduced the number gene candidates

the patient’s genome to a collection of reference genomes.

Oxford Nanopore


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Oxford Nanopore
From Oxford Nanopores Web Site

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.
!80

ytical Chemistry

•

Oxford Nanopore

REV

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).#

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
e 6. attached to the top of the nanopore to cleave single nucleotidesOxfordtarget DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace fromtheRHL protein
Biological nanopore scheme employed by from the Nanopore. (A) Schematic of RHL protein nanopore mutant depicting an positions
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
dextrin (at residue 135) and glutaminesand D reprinted with permission fromdetailed view of Natureβ barrel Group. Panels C and Enanopore shows the locati
(at residue 139). (B) A ref 91. Copyright 2009 the Publishing of the mutant reprinted with permission from Oxford
identified, as the strand remains intact. Panels A, B,
ginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave
Nanopore Technologies (Zoe McDougall).

From Niedringhaus et al. Analytical Chemistry 83: 4327. RHL protein nan
otides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an2011.
Slides single Davis EVE161 Course Taught and dCMP). (E) Winter 2014
1
hows a clear discrimination between for UC bases (dGMP, dTMP, dAMP, by Jonathan EisenStrand sequencing: ssDNA is threaded!8thro
n nanopore and individual bases are identified, as the strand Course Taught by Jonathanand D reprinted with permission from ref 91. Cop
Slides for UC Davis EVE161 remains intact. Panels A, B, Eisen Winter 2014

Analytical Chemistry

Nanopores

REVIEW

igure 5. Nanopore DNA sequencing using electronicmeasurements and optical readout as detection methods. (A) In electronic nanoporenanopo
Nanopore DNA sequencing using electronic measurements and optical readout as detection methods.(A)In electronic schemes
ignal is obtained through ionic current,73 tunneling current,78 and voltage difference79 measurements. Each method must produce a characteristic signa
schemes, signal is obtained through ionic current,73 tunneling current, and voltage difference measurements. Each method
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 the four DNAwith labeled markersoptical detected during translocation of the DNA
must produce a characteristic signal to differentiate and hybridized bases. (B) In the that are readout nanopore design, each
ragment through is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during
nucleotide the nanopore. Reprinted from ref 82. Copyright 2010 American Chemical Society.

translocation of the DNA fragment through the nanopore.
would result, in theory, inFrom Niedringhaus etthrough
detectably altered current flow al. Analyticalbilayer, using ionic current blockage method. The author
lipid Chemistry 83: 4327. 2011.
he pore. Theoretically, nanopores could also be designed to Taught by Jonathansingle nucleotides could be discriminated as lon
predicted that Eisen Winter 2014
Slides for UC Davis EVE161 Course
measure tunneling current across the pore as bases, each with a
as: (1) each nucleotide produces a unique signal signature; (2
istinct tunneling potential, could be read. The nanopore apthe nanopore possesses proper aperture geometry to accommo

Oxford Nanopore

From Oxford Nanopores Web Site

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EVE161 Lecture 2

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