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
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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: emardis@wustl.edu
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
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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
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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
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Sanger Sequencing of PhiX174
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/
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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
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From Slideshare presentation ofCosentino Cristian
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NextGen #1: 454
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NextGen #1: Roche 454
From Slideshare presentation ofCosentino Cristian
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NextGen #1: Roche 454
From Slideshare presentation ofCosentino Cristian
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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
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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
From Mardis 2008. Annual Rev. Genetics 9: 387.
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
From Mardis 2008. Annual Rev. Genetics 9: 387.
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
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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
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Ne tGen #2 Sole a
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NextGen #2: Solexa
From Slidesharepresentation ofCosentino Cristianhttp://
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N tG #2 S l Ill i
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From Slidesharepresentation ofCosentino Cristian
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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
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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
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
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 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 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
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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.
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.
Ill mina Step 3 Seq encing b S nthesis
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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...
From Mardis 2008. Annual Rev. Genetics 9: 387.
Illumina Step 3: Cycling
Illumina Evolution
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Illumina Evolution
http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
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
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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
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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
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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
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Bells and Whistles
Multiplexing
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Multiplexing
From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn
Multiplexing
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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
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Capture Methods
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Agilent SureSelect
Solution-phase capture with
streptavidin-coated magnetic beads
Reported 60-80% of capture efficiency
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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
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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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A lied Bios stems Ion Torrent PGM
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A lied Bios stems Ion Torrent PGM
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.
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
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
Single Molecule III: Oxford Nanopores
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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).
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
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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.
From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
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
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