dna sequencing -...
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![Page 1: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/1.jpg)
DNA Sequencing
Lecture3ShifraBen-Dor
![Page 2: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/2.jpg)
ClassicalMethods
• (MaxamGilbert)
• Sanger
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h?ps://binf.snipcademy.com/lessons/dna-sequencing-techniques/sanger-dideoxynucleoFde
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Whatweretheproblemsthathadtobeovercometogettohighthroughput?
• Individualsequences
• Manyatonce,butwiththeabilitytodifferenFate
• DetecFon
• Length
![Page 5: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/5.jpg)
EmulsionPCR
RadfordADetal(2012)JournalofGeneralVirology93:1853-1868
![Page 6: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/6.jpg)
BridgePCR
RadfordADetal(2012)JournalofGeneralVirology93:1853-1868
![Page 7: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/7.jpg)
RadfordADetal(2012)JournalofGeneralVirology93:1853-1868
![Page 8: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/8.jpg)
h?ps://binf.snipcademy.com/lessons/dna-sequencing-techniques/sanger-dideoxynucleoFde
![Page 9: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/9.jpg)
Shortreadtechnologies
• 454(Roche)– sequencingbysynthesis,measurebyimage(pyro)
• IonTorrent– sequencingbysynthesis,measurebypH
• SOLiD(ABI)– sequencingbyligaFon,measurebyimage
• Illumina– sequencingbysynthesis,measurebyimage
![Page 10: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/10.jpg)
WhatisNextGeneraFonSequencing?
• InWeizmann,weusetheIlluminasystem,whichisbasedonsequencingbysynthesis
• DNA(orRNA)isisolatedinthelab• DNA(orcDNA)isshearedtothepropersize• AdaptorsareaddedtotheDNA• DNAisa?achedtoaflowcell,andclustersofidenFcalsequencearegenerated
• Clustersaresequenced• Sequenceisanalyzed
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ClusterGeneraFon
• Concept:ThelibraryconsistsofmillionsofdifferentDNAmoleculeswithidenFcalends.
• EachindependentDNAmoleculewilla?achtotheflowcellandgenerateaclusterofidenFcalsequence–similartobacterialcolonies
• ClustersremaindisFnct–donotmix,sothatthesocwarecandisFnguisheachcluster
Slideprovidedby:Dr.ShirleyHorn-Saban
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SolexaSequencing
5’
3’
Primerhyb(onclusterstaFon)AddsequencingreagentsCycle1:FirstbaseincorporatedRemoveunincorporatedbasesDetectsignal
AddsequencingreagentsCycle2:SecondbaseincorporatedRemoveunincorporatedbasesDetectsignal
A
G
A T
C
G
A
T
C
G
T
C
G
AT
C
G
A
T
C
G
A
C
G A
T
C
G
A
T
CGA
T
C
CAMERA
Cleavedyeandterminator
COMPUTER
A T
CleavedyeandterminatorCycle3-n:………
T
C
T
A
C
C
A
G
Slideprovidedby:Dr.ShirleyHorn-Saban
![Page 13: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/13.jpg)
SolexaSequencing
LocaFon-Basedsequencing:
ClustersareidenFfiedbytheirlocaFonontheflowcell
Slideprovidedby:Dr.ShirleyHorn-Saban
![Page 14: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/14.jpg)
NextGeneraFon
• Illuminah?ps://www.youtube.com/watch?v=fCd6B5HRaZ8
![Page 15: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/15.jpg)
Longreadtechnologies
• PacBio
• Nanopore
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PacBio
RadfordADetal(2012)JournalofGeneralVirology93:1853-1868
![Page 17: DNA Sequencing - dors.weizmann.ac.ildors.weizmann.ac.il/course/introbioinfo/Lect3_DNAsequencing.pdfWhat is Next Generaon Sequencing? • In Weizmann, we use the Illumina system, which](https://reader033.vdocuments.us/reader033/viewer/2022041903/5e61df4847b94078e9642f6d/html5/thumbnails/17.jpg)
Nanopore
h?ps://www.youtube.com/watch?v=E9-Rm5AoZGw
PacBioh?ps://www.youtube.com/watch?v=v8p4ph2MAvI
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Roche454 EmulsionPCR Beadsandwells Pyrosequencing
SOLiD EmulsionPCR Beadsonslide LigaFon
IonTorrent EmulsionPCRIonspheresand
arrayDetecFonofreleased
H+
IlluminaBridge
amplificaFonGlassslide
hybridizaFonReversibleterminators
PacBioLinear
amplificaFon WellsFlourscentlylabeled
dNTPs
Nanopore NoamplificaFon PoresElectronicchargein
pore
SecondgeneraFon:
ThirdgeneraFon: