lectures 11, 12: dna sequencing history and...

Lectures11,12:DNASequencingHistoryandMethods

Fall2019October22,24,2019

IntroductionandHistory

SamplePreparation

SamplePreparation

Fragments

SamplePreparation

Sequencing

ACGTAGAATCGACCATG

GGGACGTAGAATACGAC

ACGTAGAATACGTAGAA

Reads

Fragments

NextGenerationSequencing(NGS)

SamplePreparation

Sequencing

Assembly

ACGTAGAATACGTAGAAACAGATTAGAGAG…

Contigs

Fragments

Reads

ACGTAGAATCGACCATG GGGACGTAGAATACGAC

ACGTAGAATACGTAGAA

SamplePreparation

Sequencing

Assembly

Analysis

Fragments

Reads

Contigs

ReferenceGenome

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Denovovs.Re-sequencing•  Denovoassembly(“fromthebeginning”)impliesthatyouhavenopriorknowledgeofthegenome.

•  Re-sequencingassemblyassumesyouhaveacopyofthereferencegenome(thathasbeenverifiedtoacertaindegree).

•  Theprogramsthatworkforre-sequencingwillnotworkfordenovo.

Denovovs.Re-sequencing

SamplePreparation

Fragments

Re-sequencing (LOCAS, Shrimp) requires 15x to 30x coverage. Anything less and re-sequencing programs will not produce results or produce questionable results.

SamplePreparation

Fragments

De-novo assembly requires higher coverage. At least 30x but upwards to 100x’s coverage. Most de novo assemblers require paired-end data.

SamplePreparation

Sequencing

Assembly

Analysis

Fragments

Reads

Contigs

Ourfocusfortoday’slecture:1.  Comparisonofsequencing

platforms2.  Detailsofsamplepreparation3.  Definitionsandterminologies

concerningdataandsequencingplatforms

HistoryandBackground

LandmarksinSequencingEfficiency(bp/person/year)

Year Event

1870 Miescher:DiscoversDNA1940 Avery:ProposesDNAas“GeneticMaterial”1953 Watson&Crick:DoubleHelixStructureofDNA

1 1965 Holley:transferRNAfromYeast1,500 1977 Maxam&Gilbert:"DNAsequencingbychemical

degradation”Sanger:“DNAsequencingwithchain-terminatinginhibitors”

15,000 1981 Messingandhiscolleaguesdeveloped“shotgunsequencing”method

25,000 1987 ABImarketsthefirstsequencingplatform,ABI370

LandmarksinSequencingEfficiency(bp/person/year)

Year Event

50,000 1990 NIHbeginslarge-scalesequencingbacteriagenomes.

200,000 1995 CraigVentureandHamiltonSmithattheInstituteforGenomicResearch(TIGR)publishedthefirstcompletegenomeofafree-livingorganisminScience.Thismarksthefirstuseofwhole-genomeshotgunsequencing,eliminatingtheneedforinitialmappingefforts.

2001 AdraftofthehumangenomewaspublishedinScience.

2001 AdraftofthehumangenomewaspublishedinNature.

50,000,000 2002 454LifeSciencescomesoutwithapyrosequencingmachine.

100,000,000 2008 Nextgenerationsequencingmachinesarrive.

Huge 2015+ OxfordNanopore:600Millionbasepairsperhour.

RobertHolleyandteamin1965

WatsonandCrick

Messing:World’smost-citedscientist

FrancisCollins:PrivateHumanGenomeproject.

Next-GenSequencingPlatforms

454/RocheGS-20/FLX(2005)

PacBioRS(2009-2010)3rdgeneration?

IlluminaHiSeq(2007)

ComparisonofPlatforms

Technology Readsperrun AverageReadLength

bpperrun Typesoferrors

454(Roche) 400,000 250-1000bp 70Million Indels

SoLiD(ABI) 88-132Million 35bp 1Billion

IlluminaHiSeq 2.5Billion 100–250bp 600Billion Substitution

PacBio 45,000 2000-10,000bp 45Million Insertionsanddeletions

\

SequencingMethodsandTerminology

Sangermethod(1977):labeledddNTPsterminateDNAcopyingatrandompoints.

Bothmethodsgeneratelabeledfragmentsofvaryinglengthsthatarefurtherelectrophoresed.

Gilbertmethod(1977):chemicalmethodtocleaveDNAatspecificpoints(G,G+A,T+C,C).

SangerSequencing

SangerSequencingVideo

SangerSequencingSHEAR DNA target sample

SangerSequencingSHEAR DNA target sample

A A A A

C GT

C GT

C GT

C GT

SangerSequencing

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SHEAR DNA target sample

A A A A

C GT

C GT

C GT

C GTA C

G

T

SangerSequencing

AC GT A

DNApolymerase Primer

SangerSequencing

AC GT A

DNApolymerase Primer

Primer

DNApolymerase

AC GT A

SangerSequencing

AC GT A

Primer

DNApolymerase

A

A

A

G

G

C

C

CT

CTA

CT

G

SangerSequencing

AC GT A

Primer

DNApolymerase

A

A

A

G

G

C

C

CT

CTA

CT

G

G

SangerSequencing

AC GT A

PrimerA

A

A

G

G

C

C

CT

CTA

CT

CG

G

SangerSequencing

AC GT A

PrimerA

A

A

G

G

C

C

CT

CTA

CT

GA

CG

G

SangerSequencing

AC GT A

PrimerA

A

A

G

G

C

C

CT

CTA

CT

T

SangerSequencing

PrimerA

A

A

G

G

C

C

CT

CTA

CT

SangerSequencing

PrimerA

A

A

G

G

C

C

CT

CTA

CT

ContinueuntilallstrandsofDNAhaveundergonethisreaction.IfyouchoosethereagentscorrectlythenyoushouldhaveallpossibleA-terminatedstrands;resultinginsequencesofvaryinglengths.

SangerSequencing

SangerSequencing

Inthegel,thelongerDNAfragmentsmovefastertothebottomandtheshorteronesmoveslowerandremainatthetop.Thesequencecanbereadoffbygoingfromtoptobottom.

Challenges

•  Requiresalotofspaceandtime:youneedaplacetorunthereaction,andthenyouneedageltodeterminethelengthoftheDNA–  Youcouldonlyrunperhapsahundredofthesereactionsatanyonetime.

–  Thereare3billionbasepairsofDNAinthehumangenome,meaningabout6million500-basepairfragmentsofDNA.

•  Nonethelessitwasstillusedtocomeupwiththefirstcopyofthehumangenome

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CeleraSequencing(2001)

•  300ABIDNAsequencingplatforms•  50productionstaff•  20,000squarefeetofwetlabspace•  1milliondollars/yearforelectricalservice•  10milliondollarsinreagents

Totalcostofhumangenome:2.7Billiondollars

CeleraSequencing(2001)

•  300ABIDNAsequencingplatforms•  50productionstaff•  20,000squarefeetofwetlabspace•  1milliondollars/yearforelectricalservice•  10milliondollarsinreagents

Currentcostofhumangenome:<10,000$

•  SecondgenerationsequencingtechniquesovercometherestrictionsbyfindingwaystosequencetheDNAwithouthavingtomoveitaround.

•  YoustickthebitofDNAyouwanttosequenceinalittledot,calledacluster,andyoudothesequencingthere;asaresult,youcanpackmanymillionsofclustersintoonemachine.

Second/NextGenerationSequencing

SequencingastrandofDNAwhilekeepingitheldinplaceistricky,andrequiresalotofcleverness.

IlluminaSequencing:Video

StepsinIlluminasequencing

•  Turnonthesequencingmachineandwait(1week)…

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StepsinIlluminasequencing

•  Sampleprep:sizeselectfragments,addadapterstoensurethefragmentsligatetotheflowcell(1to5days)

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ligateadapters

StepsinIlluminasequencing•  Clustergenerationonflowcell

Whydoweneedclusters?

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Aflowcellcontains8lanes

Eachlanecontainsthreecolumnsoftiles

Eachcolumncontains100tiles

20Kto30KclustersEachtileisimagedfourtimespercycle,whichisoneimageperbase

Wemultiplyupthetemplatestand,i.e.thebitofDNAthatwearesequencing,andstickonafewbasesof‘adaptorsequence’;thissequencesticksontocomplementarybitsofDNAstucktoasurface,whichholdstheDNAinplacewhilewesequenceit:

WethenfloodtheDNAwithReversibleTerminator(RT)-bases.Wealsoaddapolymeraseenzyme,whichincorporatestheRT-baseintothenewstrandthatiscomplementarytothetemplatestrand:

WethenwashawayalltheRT-bases,leavingjustthosethatwereincorporatedintothenewstrand;wecanreadoffwhatbasethisisbylookingatthecolorofthedye:

There exists a cleavage enzyme thatchops all the extramolecules off, andturns the RT-base into a normallyfunctioningnucleotide.

•  IlluminausesthemodifiedversionofSangersequencingcalledreversibleterminatormethod.

•  Thedyeiswashedafterimagingandthelastnucleotideisextendedinthenextround.

•  InasingleIlluminamachinewehavehundredsofmillionsoftheseclusters;cameraslookatallofthesedotsandrecordhowtheychangecolorovertime,allowingyoutodeterminethesequenceofbasesofmillionsofbitsofDNAatonce.

IlluminaCharacteristics

•  Sequencingmethodisactuallyprettyinefficient,however,themachineiscapableofsequencingmillionsoffragmentsofDNAatonce.

•  Duetocontrolledsequenceoftermination,washing,andchemicaldeactivation/activationevents,Illuminareadshave(almost)onlysubstitutionerrors.

•  Pairedreadswithsmallinsertsize(<800bp)canbereliablygenerated.Largeinsertmatepairscanbemadeusingunreliable,difficult,time-consuming,andexpensivechemicalhacks.

IlluminaCharacteristics

InsidetheIlluminaMachine

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Pyrosequencing:Video454RocheSystem

https://www.youtube.com/watch?v=nFfgWGFe0aA

•  PyrosequencingdiffersfromSangersequencing,inthatitreliesonthedetectionofpyrophosphatereleaseonnucleotideincorporation,ratherthanchainterminationwithdideoxynucleotides.

•  Sincethereisnochainterminationinpyrosequencingotherthanbydesignedunavailabilityoftheother3nucleotides,pyrosequencingreadshaveinsertion/deletionerrorsparticularlyinornexttorunsofhomopolymers:hardtodistinguishbetweenAAAAAandAAAAAA

PyrosequencingCharacteristics

•  Relativelylongreads:800-1000bp.

•  Reliablepairedreadprotocolwithlargeinsertsizes:3kbp,8kbp,20kbp.

•  Forinstance,apairof1000bpreadsbacktoback(insertsize=2kbp)essentiallygivesa2000bpread.

•  Dealingwith2%-3%indelsin454readsisthemainchallengebesidehighersequencingcostsincomparisonwithIllumina.

PyrosequencingCharacteristics

SingleMoleculeSequencing:VideoPacificBiosciencesSystem

https://www.youtube.com/watch?v=v8p4ph2MAvIhttps://www.youtube.com/watch?v=NHCJ8PtYCFc

•  PacBioreadsarelong,e.g.onaverageafewkilobases.

•  SincePacBioreliesonthesignalfromasinglemolecule,thesignaltonoiseratioissmall,andPacBioreadshavelotsofuniformlyrandomerrors,upto15%.

•  PacBioerrorsareprimarilyindels,whichmakesefficaciouscomputationalerrorcorrectioncurrentlyintractable.

•  PacBioreadsarecurrentlyusedforlimitedvalidationofcontiguityinformationorhelpingdatasetsgeneratedwithothertechnologies.

PacBioCharacteristics

NanoporeSequencing:VideoOxfordNanoporeSystemhttps://www.youtube.com/watch?v=3UHw22hBpAk

•  Nanoporereadsareprettylong,upto100+kbp.

•  Theyhavelotsoferrors,10%-40%.

•  ErrorsareprimarilyindelslikePacBio’sbuttheNanoporeerrormodelisnotclearyet[PacBioerrorsareprettymuchuniformlyrandom].

•  Nanoporehasjuststartedaworld-widebenchmarkingprojectwhichisstillgoingon.

OxfordNanoporeCharacteristics

lectures 11, 12: dna sequencing history and...

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