History of DNA sequencing

7/23/2019 History of DNA sequencing

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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics

Evolution of Sequencing

Workshop in Applied PhylogeneticsMarch 9, 2014Bodega Bay Marine Lab

Jonathan A. Eisen

UC Davis Genome Center
http://phylogenomics.wordpress.com/


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Review Papers

Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem2013;6:287-303. doi: 10.1146/annurev-anchem-062012-092628.
http://www.ncbi.nlm.nih.gov/pubmed?term=Mardis%20ER%5BAuthor%5D&cauthor=true&cauthor_uid=23560931http://phylogenomics.wordpress.com/


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Review Papers

Next-Generation DNASequencing Methods

Elaine R. Mardis

Departments of Genetics and Molecular Microbiology and Genome Sequencing Center,Washington University School of Medicine, St. Louis MO 63108; email: [email protected]

Annu. Rev. Genomics Hum. Genet. 2008.

9:387402


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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
http://m.cancerpreventionresearch.aacrjournals.org/content/5/7/887.fullhttp://phylogenomics.wordpress.com/


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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 HarbSymp 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: 86092; Science, 2001, 291: 13041351)

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

MiseqRoche JrIon TorrentPacBioOxford

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

Sequencing Technology Timeline
http://www.slideshare.net/cosentia/high-throughput-equencinghttp://phylogenomics.wordpress.com/


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Generation I: Manual Sequencing


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Maxam-Gilbert Sequencing

http://www.pnas.org/content/74/2/560.full.pdf
http://www.pnas.org/content/74/2/560.full.pdfhttp://phylogenomics.wordpress.com/


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Sanger Sequencing of PhiX174

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


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Sanger Sequencing


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Sanger Sequencing


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Nobel Prize 1980: Berg, Gilbert, Sanger


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Generation II: Automated Sanger


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Automation of Sanger Part I


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Automation of Sanger Part II


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Automated Sanger Highlights

1991: ESTs by Venter 1995: Haemophilus influenzaegenome

1996: Yeast, archaeal genomes

1999: Drosophilagenome 2000:Arabidopsisgenome

2000: Human genome

2004: Shotgun metagenomics


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Generation III: Clusters not Clones


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Generation III = NextGen


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NextGen Sequencing Outline

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

Amplification

Sequencing

Imaging

Four colour imaging

Data analysis

!"#$% '(')**+,-./ 01)) 12- 345-6

From Slideshare presentation ofCosentino Cristian

http://www.slideshare.net/cosentia/high-throughput-equencing


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NextGen #1: 454


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NextGen #1: Roche 454




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NextGen #1: Roche 454




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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
http://acb.qfab.org/acb/ws09/presentations/Day1_DMiller.pdfhttp://phylogenomics.wordpress.com/


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DNA library preparation

A

A

A B

B

B

4.5 hours

Ligation

Selection

(isolate AB

fragments

only)

Genome fragmentedby nebulization

No cloning; no colony picking

sstDNA library createdwith adaptors

A/B fragments selectedusing avidin-biotin purication

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 Ste 1: Libraries


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Anneal sstDNA to an excess ofDNA capture beads

Emulsify beads and PCRreagents in water-in-oil

microreactors

Clonal amplication occursinside microreactors

Break microreactors andenrich for DNA-positive

beads

Emulsion PCR

8 hours

sstDNA library Bead-amplied sstDNA library


Roche 454 Ste 2: Emulsion PCR

R h 454 St 3 P i


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Amplied sstDNA library beads Quality ltered bases

Sequencing

7.5 hours

Well diameter: average of 44 m

400,000 reads obtained in parallel

A single cloned amplied sstDNA bead is deposited per well


Roche 454 Step 3: Pyrosequencing

R h 454 St 3 P i


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44 m

Pyrosequecning

Reads are recorded as flowgrams

Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402

Nature Reviews genetics, 2010, 11: 31-46

From Slidesharepresentation of CosentinoCristianhttp://www.slideshare.net/cosentia/high-throughput-equencing

Roche 454 Step 3: Pyrosequencing

R h 454 K I


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Roche 454 Key Issues

Number of repeated nucleotidesestimated by amount of light ... manyerrors

Reasonable number of failures in EM-PCR and other steps

R h 454 E l ti


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Roche 454 Evolution


Ne tGen #2 Sole a


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NextGen #2: Solexa

From Slidesharepresentation ofCosentino Cristianhttp://

www.slideshare.net/cosentia/high-throughput-equencing

N tG #2 S l Ill i


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From Slidesharepresentation ofCosentino Cristian


NextGen #2: Solexa Illumina

NextGen #2: Illumina Accessories


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NextGen #2: Illumina Accessories

Cluster station

Genome Analyzer IIxPaired-end module Linux server

Bioanalyzer 2100

From Slidesharepresentation ofCosentinoCristianhttp://www.slideshare.net/cosentia/high-throughput-equencing

Illumina Outline


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Illumina Outline

Clusters

amplification

Clusterstation

Wash clusterstation

Clustergeneration

Linearization,

Blocking and

primer

Hybridization

Read 1

Prepare read 2

Read 2GAIIx&P

E

SBSsequencing

Pipeline base call

Data analysis

Sample

preparation and

library validation

Analysis

From Slidesharepresentation ofCosentino Cristianhttp://www.slideshare.net/cosentia/high-throughput-equencing

Ill i St 1 P & Att h DNA


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Illumina Ste 1: Pre & Attach DNA

Adapter

DNA fragment

Dense lawnof primers

Adapter

DNA

Adapters

Prepare genomic DNA sample

Randomly fragment genomic DNAand ligate adapters to both ends ofthe fragments.

Attach DNA to surface

Bind single-stranded fragmentsrandomly to the inside surfaceof the ow cell channels.

a


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

Illumina Step 2: Clusters by Bridge PCR


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Illumina Step 2: Clusters by Bridge PCR

Attached

Bridge amplication

Add unlabeled nucleotides

and enzyme to initiate solid-

phase bridge amplication.

Denature the doublestranded molecules

Nucleotides


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 whic

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 andextension 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.

Clusters
http://seqanswers.com/forums/showthread.php?t=21http://phylogenomics.wordpress.com/


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Clusters

Ill i St 3 S i b S th i


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b

Laser

First chemistry cycle:determine rst base

To initiate the rst

sequencing cycle, add

all four labeled reversible

terminators, primers, and

DNA polymerase enzyme

to the ow cell.

Image of rst chemistry cycle

After laser excitation, capture the image

of emitted uorescence from each

cluster on the ow cell. Record the

identity of the rst base for each cluster.

Before initiating thenext chemistry cycle

The blocked 3' terminus

and the uorophore

from each incorporated

base are removed.


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.

Ill mina Step 3 Seq encing b S nthesis
http://seqanswers.com/forums/showthread.php?t=21http://phylogenomics.wordpress.com/


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Illumina Step 3: Sequencing by Synthesis

Illumina Step 3: Cycling


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Sequence read over multiple chemistry cycles

Repeat cycles of sequencing to determine the sequenceof bases in a given fragment a single base at a time.

GCTGA...


Illumina Step 3: Cycling

Illumina Evolution


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Illumina Evolution


MiSeq Dx


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MiSeq Dx

HiSeq x Ten


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HiSeq x Ten

HiSeq x Ten


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HiSeq x Ten

NextGen #3: 454: ABI Solid


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NextGen #3: 454: ABI Solid


ABI Solid Details


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ABI Solid Details

A C G T1stbase

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 (n1)

Ligase

Phosphatase

+

1. Prime and ligate

2. Image

4. Cleave offuor

5. Repeat steps 14 to extend sequence

3'

Universal seq primer (n1)1.Melt offextendedsequence

2.Primer reset3'

AA AC G

G GG

C C

C

T AA

A GG

CC

T TTT

6. Primer reset

7. Repeat steps 15 with new primer

TA

AT

AT3'

TA

AT3'

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 mbead

1 mbead

1 mbead

1

i l i

Primer round 1

Template

Primer round 2 1 base shift

Glass slide

3'5' Template sequence

1mbead

P1 adapter

a

The ligase-mediated sequencing approach of theApplied Biosystems SOLiD sequencer. In amanner similar to Roche/454 emulsion PCR

amplification, DNA fragments for SOLiDsequencing are amplified on the surfaces of 1-!m

magnetic beads to provide sufficient signal duringthe sequencing reactions, and are then depositedonto a flow cell slide. Ligase-mediatedsequencing begins by annealing a primer to theshared adapter sequences on each amplifiedfragment, and then DNA ligase is provided alongwith specific fluorescent- labeled 8mers, whose4th and 5th bases are encoded by the attached

fluorescent group. Each ligation step is followedby fluorescence detection, after which aregeneration step removes bases from theligated 8mer (including the fluorescent group)and concomitantly prepares the extended primerfor another round of ligation. (b) Principles of two-base encoding. Because each fluorescent groupon a ligated 8mer identifies a two-basecombination, the resulting sequence reads canbe screened for base-calling errors versus truepolymorphisms versus single base deletions by

aligning the individual reads to a known high-quality reference sequence.

From Mardis 2008. Annual Rev.Genetics 9: 387.

ABI Solid Evolution


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ABI Solid Evolution


Complete Genomics


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Complete Genomics

Figure 3. Schematic of Complete GenomicsDNB 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 probescorresponding 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.

Comparison in 2008


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Comparison in 2008

From Introduction to Next Generation Sequencing by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/

CSHL_nextgen.ppt

Comparison in 2012
http://prometheus.cshl.org/twiki/pub/Main/CdAtA08/CSHL_nextgen.ppthttp://phylogenomics.wordpress.com/


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Comparison in 2012

From Introduction to Next Generation Sequencing by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/

CSHL_nextgen.ppt

Bells and Whistles
http://prometheus.cshl.org/twiki/pub/Main/CdAtA08/CSHL_nextgen.ppthttp://phylogenomics.wordpress.com/


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Bells and Whistles

Multiplexing


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Multiplexing

From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn

Multiplexing
http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmnhttp://phylogenomics.wordpress.com/


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Multiplexing

http://res.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf

Small Amounts of DNA


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Small Amounts of DNA

http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4

Capture Methods


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Capture Methods

-

RainDance

Microdroplet PCR

Roche Nimblegen

Salid-phase capture with custom-

designed oligonucleotide microarray

Reported 84% ofcapture efficiency

Reported 65-90% of capture efficiency

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

Capture Methods


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Agilent SureSelect

Solution-phase capture with

streptavidin-coated magnetic beads

Reported 60-80% of capture efficiency

From Slideshare presentation ofCosentino Cristianhttp://www.slideshare.net/cosentia/high-throughput-equencing

Capture Methods

Illumina Paired Ends


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Illumina Paired Ends

Paired-end sequencing works into GA and uses chemicals from the PE

module to perform cluster amplification of the reverse strand


Moleculo


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Moleculo

Large fragmentsDNA

Isolate and amplify

CACC GGAA TCTC ACGT AAGG GATC AAAA

Sublibrary w/ unique barcodes

Sequence w/ Illumina

Assemble seqs w/ same codes

Generation III+: Faster w/ Clusters


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Generation III : Faster w/ Clusters

Ion Torrent PGM


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A lied Bios stems Ion Torrent PGM


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Workflow similar tothat for Roche/454systems.

Not surprising,since invented bypeople from 454.

Ion Torrent pH Based Sequencing


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p q g

Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem2013;6:287-303.

Ion Torrent Evolution


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Generation IV: Single Molecule


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Generation IV: Single Molecule

Sin le Molecule I: Helicos


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4+$0510#

Single Molecule II: Pacific Biosciences


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g

Sin le Molecule II: Pacific Biosciences


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Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem2013;6:287-303.

Sin le Molecule II: Pacific Biosciences


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Figure 2. Schematic of PacBios real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single

DNA polymerase (!29) 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.


Why Finish Genomes?


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Why Finish Genomes


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JOURNAL OFBACTERIOLOGY, Dec. 2002, p. 64036405 Vol. 184, No. 230021-9193/02/$04.000 DOI: 10.1128/JB.184.23.64036405.2002

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

http://jb.asm.org/content/184/23/6403.full

HGAP Assembly from PacBio


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HGAP Assembler for PacBio Data

http://www.pacificbiosciences.com/pdf/

microbial_primer.pdf

Detecting Modified Bases


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Figure 2. Principle of detecting modified DNA bases during SMRTsequencing. 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. longerinterpulse duration (IPD), compared to a control DNA template lacking the modification (bottom).

3

http://www.pacificbiosciences.com/pdf/microbial_primer.pdf

Single Molecule III: Oxford Nanopores


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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 thismembrane. 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 themolecule 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 identifytarget 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



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Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore.

(A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue139). (B) A detailed view of the "barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and thecysteines. (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 "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 a

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 Oxfor

Nanopore Technologies (Zoe McDougall).




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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 differencemeasurements. 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.


Oxford Nanopores MinIon


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Its 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 ismixed. From http://www.nature.com/news/data-from-pocket-sized-genome-sequencer-unveiled-1.14724
http://www.nature.com/news/data-from-pocket-sized-genome-sequencer-unveiled-1.14724http://phylogenomics.wordpress.com/


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Documents

History of DNA sequencing