DNA Sequencing

Lecture3ShifraBen-Dor

ClassicalMethods

•  (MaxamGilbert)

•  Sanger

h?ps://binf.snipcademy.com/lessons/dna-sequencing-techniques/sanger-dideoxynucleoFde

Whatweretheproblemsthathadtobeovercometogettohighthroughput?

•  Individualsequences

•  Manyatonce,butwiththeabilitytodifferenFate

•  DetecFon

•  Length

EmulsionPCR

RadfordADetal(2012)JournalofGeneralVirology93:1853-1868

BridgePCR

RadfordADetal(2012)JournalofGeneralVirology93:1853-1868

RadfordADetal(2012)JournalofGeneralVirology93:1853-1868

h?ps://binf.snipcademy.com/lessons/dna-sequencing-techniques/sanger-dideoxynucleoFde

Shortreadtechnologies

•  454(Roche)– sequencingbysynthesis,measurebyimage(pyro)

•  IonTorrent– sequencingbysynthesis,measurebypH

•  SOLiD(ABI)– sequencingbyligaFon,measurebyimage

•  Illumina– sequencingbysynthesis,measurebyimage

WhatisNextGeneraFonSequencing?

•  InWeizmann,weusetheIlluminasystem,whichisbasedonsequencingbysynthesis

•  DNA(orRNA)isisolatedinthelab•  DNA(orcDNA)isshearedtothepropersize•  AdaptorsareaddedtotheDNA•  DNAisa?achedtoaflowcell,andclustersofidenFcalsequencearegenerated

•  Clustersaresequenced•  Sequenceisanalyzed

ClusterGeneraFon

•  Concept:ThelibraryconsistsofmillionsofdifferentDNAmoleculeswithidenFcalends.

•  EachindependentDNAmoleculewilla?achtotheflowcellandgenerateaclusterofidenFcalsequence–similartobacterialcolonies

•  ClustersremaindisFnct–donotmix,sothatthesocwarecandisFnguisheachcluster

Slideprovidedby:Dr.ShirleyHorn-Saban

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

SolexaSequencing

LocaFon-Basedsequencing:

ClustersareidenFfiedbytheirlocaFonontheflowcell

Slideprovidedby:Dr.ShirleyHorn-Saban

NextGeneraFon

•  Illuminah?ps://www.youtube.com/watch?v=fCd6B5HRaZ8

Longreadtechnologies

•  PacBio

•  Nanopore

PacBio

RadfordADetal(2012)JournalofGeneralVirology93:1853-1868

Nanopore

h?ps://www.youtube.com/watch?v=E9-Rm5AoZGw

PacBioh?ps://www.youtube.com/watch?v=v8p4ph2MAvI

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: