3/11/2015 WhatssospecialaboutNextGenerationsequencing?OxbridgeBiotechRoundtable

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WhatssospecialaboutNextGenerationsequencing?

Sunday,19thAugust2012PolicyPulse

NextGenerationDNAsequencingtechnologyisprogressingrapidly,producingconsiderableadvancementsindiversefieldsrangingfromretracingthestepsofa5,000yearoldIcemantodevelopingbreastcancertherapies.AsearchforNextGenerationSequencinginthePubMeddatabaserevealsover1,000paperspublishedinthelastyearalone,withmanygeneticsstudiesreachingmainstreamnewssitessuchastheBBC,GuardianScienceandtheTelegraph.Withthesenewtechnologiesawiderangeofscienceisnowfeasiblethatwaspreviouslyunimaginable.Wholegenomesequencingofthe5,000yearoldIcemantzi,foundintheFrenchAlpsin1991,revealedageneticpredispositiontoheartdiseaseandvaluablehistoricalinformationabouttzisorigin,bloodtypeandcluestohisappearance(1,2).TheHumanMicrobiomeProjectaimstosequenceallthebacteriaandmicrobesinthehumanbody(3)improvingdiagnosis,treatmentandunderstandingofanarrayofdiseases.Sequencingofcancergenomesisrevealingawidegeneticvariationwithinasingletumour,withimplicationsfordrugresistanceandtreatmentofthedisease(46).TheCancerGenomicsHubwasannouncedinCaliforniainMaythisyearacollectionofallthedatafromthethreebigsequencingcentresintheUSA,holding5petabytesofinformationoncancergenomics(7,8).

Whiletheseandotherprojectsadvancerapidly,theyaredependentonthetechnologyandhowgovernments,doctorsandscientistsarechoosingtouseit.Earlierthisyear,theannouncementbyOxfordNanoporeTechnologies(9)ofaUSBsizedmachineinwhichsequencingcanbecarriedoutinaslittleas15minuteswithanexpectedretailofaround$900willhaveimportantimplicationsinbothacademicandmedicallabs.Pavingtheway,Norwayannouncedthis

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3/11/2015 WhatssospecialaboutNextGenerationsequencing?OxbridgeBiotechRoundtable

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yearthatNextGenerationSequencingwillbeincludedinitshealthserviceforthetreatmentofcancer(10),whilemanyothercountriesareusingNextGenerationTechnologiesinresearchhospitals.Whiletheseadvancementssoundspectacular,theyhavecomeafterthehardworkofgreatscientistsofanearlierera,andmuchworkremainstoovercomethelingeringhurdlesofthescienceandalsotheregulationofNextGenerationsequencing.

Lettherebelight

WhileNextGenerationSequencingtechnologiesallowforthesequencingofawholegenomeinjustafewdays,originalsequencingtechnologiesofteninvolveddangerousradiationandtedioustimeconsumingsteps.

Thefirstbigbreakthroughsinsequencingtechnologieshappenedinthemid1970s,withaseriesofmethodsbasedonpolyacrylamidegelelectrophoresis,whichallowsDNAfragmentstobedistinguishedbytheirsize.FrederickSangersinitialplusandminussequencingmethod,publishedin1975,wasthefirstofthese.Itwasdependentoncomparingthelengthsofnucleotidesextendedinthepresence(plus)orabsence(minus)ofaparticularnucleicacid(A,C,T,orG).Whilethiswasquiteanadvance,thismethodhadlimitations:itcouldonlydetermine50basesperreaction,notallbasesinthemiddleoftheruncouldbedetermined,anditwasdifficulttodeterminethelengthofthesequencingruns(11).

Twomethodspublishedin1977tookthisconceptevenfurther.ThefirstofthesewasMaxamGilbertsequencing,orChemicalSequencing,developedbyAllanMaxamandWalterGilbert,andwasthefirstwidelyusedsequencingmethod.TheprocessusedradioactivelabelingofdoublestrandedDNAfragments.TheDNAwasthencleavedbybasespecificchemicalreactionsandthefragmentsseparatedbyelectrophoresis(Fig.1a).ThesecondmethodwasFrederickSangersimprovementonhisplusandminusmethodwiththedideoxyorchainterminationmethod.Theprocessisstillwidelyusedtoday.InsteadofchemicalcleavageoftheDNA,theprocessdependson32Plabelledchainterminatingdideoxynucleotides,whichpreventfurtherextensionofthesequenceuponincorporation[Fig.1b].Eachreactionthusgeneratesfragmentsofincreasingsize,endingatthebasespecifiedbythereactioni.e.eachA,T,CorG.Thismethodoriginallyallowedreadingofsequencesupto100basepairslong(11).SangerandGilbertreceivedtheNobelPrizeforchemistryin1980fortheircontributiontoDNAsequencingtechnologies,sharedwithPaulBergforhisworkonthechemistryofDNAandrecombinantDNA(12).Improvementsinthegeltechnologyandbettergelresolution,duetoradioactivelabellingwith35Sorfluorescenttagsratherthan32P,allowedsequencingofupto30,000basesofDNAinoneday,withuptoapproximately400basessequencedperreactionbytheearly1980s(11).

Figure1TheOriginalSequencingTechnologiesA)MaxamGilbert(Chemicalsequencing)andB)SangerDideoxyChainterminationsequencing.Part3showsDNAfragmentsresolvedonapolyacrylamidegelandasequencingtracefromamodernautomatedsequencingmachine.FiguresbySarahEtheridge

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3/11/2015 WhatssospecialaboutNextGenerationsequencing?OxbridgeBiotechRoundtable

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Withproductionofsuchlargequantitiesofsequencingdata,thedataprocessingsoonbecamearesearchtopicinitselfandthebirthofbioinformaticswasinevitable.Thenin1986,encouragingtheriseofthisnewfield,LeroyHoodatCaltech,incollaborationwithAppliedBiosystems(ABI),publishedthefirstreportofsequencingdatabeingcollecteddirectlytoacomputer.Thetechnology,basedonSangersdideoxymethod,usessequencingprimersfluorescentlyendlabelledwithfourdifferentcolourstorepresenteachbase.Reactionsarethenrunsimultaneouslythroughapolyacrylamidetubegel,withtheDNArecognisedbyitsfluorescenceasitpassesadetector.AseriesofnewandimprovedABImachineswerereleasedinthefollowingyearswithdedicatedsequencingfacilitiessetupwiththeeventualaimofsequencingthehumangenome(11).

Sequencingjustgotbetter

DiscussionsabouttheHumanGenomeProjectofficiallybeganatameetingin1985,witha5yearplanpublishedtotheDOEandNIHin1990.AlthoughbeginningintheUS,theprojectbecameaninternationalcollaborationbetweencentresintheUS,EuropeandJapanwitheachcentrefocusingonparticularregionsofthegenome.In1992,CraigVenterandcolleaguesattheNIHsetuponeofthefirstdedicatedsequencingfacilitiesTheInstituteforGenomicResearchTIGR.

Figure2WholeGenomeShotgunsequencing(WGS)Adaptedfrom:http://www.bio.davidson.edu/courses/genomics/method/shotgun.htmlandhttp://www.biomedcentral.com/14712164/11/438/figure/F1?highres=y

In1995,hisgroupandcollaboratorsreportedthecompletegenomesequencesofthebacteriaHaemophilusinfluenzaeandMycoplasmagenitalium,thelargestgenomicsequencespublishedatthattime.VenterandhisteamintroducedcriticalimprovementstoamethodknownastheWholeGenomeShotgun(WGS)approach,whichlaterformedthebasisofNextGenerationSequencing.InWGS,wholegenomicDNAisrandomlyfragmentedandclonedintoE.Coli[Fig.2].Theclonesaresequencedatrandomandassembledwithspecialisedsoftware.TheTIGRassemblerwasoneofthefirstpiecesofsoftwarethatallowedtheanalysisofthousandsofsequencereadsmakinginterpretationofthedatapossible.

ThedevelopmentoftheABI3700Capillarysequencerinthelate1990swasanotherkeyprogressionduringthistime,

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allowingsimultaneoussequencingofupto96samplesthroughseparatecapillariesfilledwithnoncrosslinkedpolymermatrix.CraigVenterandcolleaguesadoptedtheABI3700withtheircompanyCelera,whichworkedindirectcompetitiontothepublicHumanGenomeproject.Theresultsofeachprojectwerepublishedinthesameweek,withthefirstdraftsequencesoftheHumanGenomeProjectpublishedinNatureandSciencein2001.

WhiletheWGSapproachledtothedevelopmentoftheNextGenerationSequencingmethods,whichreadhundredsorthousandsofsequencesinparallel,thehighthroughputtechnologycancompromisetheaccuracyandlengthofthesequencessuchthatfollowupwithdideoxySangersequencingisoftennecessary.ExomeSequencing,alsoknownasTargetedExomeCapture,isavariationofNextGenerationSequencingthatonlysequencesDNAregionsthatencodeproteins,knownasexons.Exonsonlyaccountforaround1%ofthegenome,sothismethodallowstheexclusionofthelargeintronicsequencesfoundwithinandbetweengenes,thussavingtimeandreducingcost.Thismethoddoesriskexcludingmutationsinnoncodingregionsofthegenome,however,andseveraldiseaseshaverecentlybeenlinkedtomutationsintheseareas.

SomeofthefirstcommerciallyavailableNextGenerationtechnologiesweredevelopedby454LifeSciencesandSolexatechnology,laterpurchasedbyIllumina.BothmethodsinvolverandomshearingofgenomicDNA,followedbylinkingtobeadsoraspecialisedslide.The454LifeSciencessystem[Fig.3A]isbasedonthepyrosequencingmethod,whichallowsshotgunsequencingwithoutcloninganyoftheDNA.PyrosequencinginvolvesaDNAsynthesisreaction,witheachofthefourdNTPbasesappliedoneafteranother.DuringaDNAsynthesisreaction,aPhosphategroupisreleasedwhentwonucleotidesarebound.PyrosequencingmeasurestheamountofphosphatereleasedaseachdNTPisaddedtothereactionandincorporatedintothesequence(13).Drawbacksincludetheriskofbasesbeingfalselyinsertedorremoved/deletedfromthesequenceduetomisjudgmentsinthelengthofthesequencingrun.TheseerrorsareknownasIndels.Themethodcanproduceonemillionbasesofsequencewith99.5%accuracy,witheachreadmorethan250baseslong(13).Solexatechnology[Fig.3B]alsousesDNAsynthesisreactionsbutmeasuringfluorescenceratherthanpyrophosphatereselase.Fluorescentlylabeledchainterminatingnucleotidesareincorporatedintothesequenceandmeasuredbyadetector.However,theincorporationofthechainterminatingnucleotideisreversible,allowingthesynthesistocontinueuntilanotherchainterminatingnucleotideisincorporated,sothebasesineachsequencearemeasuredoneatatime.Aseachbaseofthesequenceisreadinanindividualstep,thenumberofindelsisreducedcomparedtothe454technology.Reversibledyeterminatornucleotidesarenotincorporatedefficiently,meaningasmallerreadlengthcomparedtothe454andmorebasesubstitutionerrors.Themethodcanproduce1billionbasesof3040basesequencesinasinglerun(14).

Figure3NextGenerationSequencingMethodsA)454Methodadaptedfrom(13)http://www.wellcome.ac.uk/Educationresources/Teachingandeducation/Animations/DNA/WTX056046.htm

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B)SolexaTechnology,adaptedfrom(14)http://wellcome.ac.uk/Educationresources/Teachingandeducation/Animations/DNA/WTX056051.htm

Asdescribedabove,theannouncementofthereleaseoftheNanoporeSequencingtechnologyisanexcitingdevelopment,withthepotentialtorevolutionisethesequencingworldandgenomicmedicine.Thetechnologyisremarkablysmart:DNAnucleotidesareguidedthroughnanoporesbyanenzyme,interruptingtheflowofionsthroughtheporewhentheypassthrough.Eachnucleotidecanthusbedetectedasadistinctelectricalsignalduetodifferencesintheinterruptionofionflow.LongstrandsofDNAcanbereadnucleotidebynucleotideinthismanner[Fig.4].Furtherdetailsofthetechnologyanditscompetitioncanbefoundhere.NanoporetechnologyrepresentsarapidstepforwardbecausenomodificationoramplificationoftheDNAisneededbeforesequencingtakesplace,savingtimeandreducingerrorssosequencingcanbecarriedoutinaslittleas15minutes.TheportableMinIONdevicewouldallowdoctorstosequencedirectlyfromapatientsbloodintheclinic,whilethelargerGridIONdevicecansequenceanentiregenomeinaday.Thelowoperatingandretailcostsalsomeanthatthegoalofthelongawaited$1,000dollargenomehasnowbeenreached,makinggenomicmedicinearealistictreatmentoptionformanypatientsinthenearfuture(1517).

Figure4NanoporeSequencingTechnologyProteinnanoporesaresetinanelectricallyresistantmembranebilayer.Anioniccurrentispassedthroughthenanopores,andifananalytepassesthroughtheporeornearitsaperture,acharacteristicdisruptionofcurrentiscreated.Bymeasuringthatcurrent,itispossibletoidentifythemoleculeinquestion.Duringsequencing,forexample,aDNAstrandisfedthroughthenanoporebyanenzymeandeachofthefourstandardDNAbasesG,A,TandCcanbeidentified.From(16)http://www.nanoporetech.com/technology/introductiontonanoporesensing/introductiontonanoporesensing

Anethicalnomansland

Withtherapidlydecreasingcostandtimeofgenomesequencingcomeseverallegalandethicalissues.Ownershipofthesequencingdata,storage,accessandprivacyaresignificantconcernsoncethepatientssamplehasbeentaken.Afearofgeneticdiscrimination,forexamplebyinsurancecompanies,mayalsopreventpeopleundergoinggenetictestingwhenitwouldbeofbenefit(1820).Ownershipofdonatedsamplesbecomesaparticularissuewhenthesamplesproducescientificbreakthroughsofusetothebiotechindustry.OnefamousexampleofcontroversialuseofapatienttissueisHeLacells,thefirstimmortalisedcellline,whichwereisolatedfromacervicalcancerbiopsytakenwithoutconsentfromthepatientHenriettaLacksinthe1950s(21).

ArelatedcaseinvolvinggeneticinformationisthatofGreenbergv.MiamiChildrensResearchInstitute(2003).TheplaintiffsinthiscasewereagroupoffamiliesaffectedbyCanavandisease,aninheriteddegenerativebrainconditionresultingfromaninabilitytoprocesstheaminoacidAsparticacid.ThefamilieshadpreviouslypersuadedDrReubenMatalontoconductresearchtoidentifythegeneresponsibleforthediseaseandhadsetupatissuebankwiththeaimoffindinganaffordablediagnostictest.Theydidindeedfindthegeneanddevelopatest.However,Matalonwentontoobtainapatentforthegenewithoutthefamiliesconsent,sohisemployer,MiamiChildrensResearchInstitute,gainedcontroloftestingforthedisease.Thefamiliesbelievedthattheseactionsdisagreedwiththeoriginalpurposeofthedonations.Thecourtdidnotfindthatthefamilieshadapropertyrighttothetissue,however,statingthatthepropertyrightinbloodandtissuesamplesevaporatesoncethesampleisvoluntarilygiventoathirdparty(22,23)

Thegeneralfeelingbythecourtsappearedtobethatthereisadangerifpeoplecanexploittheirbodiesforfinancialgain,andthatthismaynegativelyimpactonthebiotechindustryandthereforeonthedevelopmentofresearchand

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healthcare.Somearguefortissuedonorstobenamedasinventorsonpatentsarisingfromtheirtissueasuniquematerial,whichmayincentivisetissuedonation(23).Theargumentagainstthisisthatapatentrequiresaninventivestepandthereforedetailedknowledgeofhowtoadvancetheresearchusingthedonatedsample..TheargumentislessthanclearwiththeGreenbergcase,asthefamilieswishedtheirtissuetobeusedforresearch.However,theymaynothavetheknowledgetoconducttheexperimentsneededtoidentifythegeneanddevelopthediagnosticprocedures.Aninnovativemother,SharonTerry,whosetwochildrenareaffectedbytheinheritedconditionpseudoxanthomaelasticum(PXE)wasnamedascoinventorforthepatentforthePXEgene(ABCC6)whichaffectsconnectivetissueintheskin,retinaandthecardiovascularsystem.ShefoundedPXEinternationalwithherhusband,settingupacentralizedtissuebankandcoordinatinggenetic,epidemiologicalandotherstudies.MrsTerryisalsonowCEOoftheGeneticAlliance(2325).

Personalisedmedicineandthefuture

Sequencingtechnologyhasadvancedmassivelysinceitsbirthinthe1970s,withmanytechnologiesreleasedthisyearpotentiallyallowingsequencingofwholegenomesinadayforlessthan$1,000,agoalthatcouldonlybedreamedaboutafewyearsago.

Thetechnologicaldevelopmentsraiseimportantethicalissues,whichareslowlybeingaddressed.Rapidinterpretationofthemassesofdataproducedcurrentlyrequireshighlyspecializedsoftware,andrepresentsoneofthenextchallengestobringingwholegenomesequencingroutinelytotheclinic.Theareaofpharmacogenomicslookingatgeneticcombinationsorbarcodesofanindividualfortargetingtherapy,asopposedtohominginonaspecificgeneislikelytobeanimportantfocusfordeterminingpeopleatriskofcommondiseases.Identificationofthe10typesofbreastcancerearlierthisyearrepresentsanexampleofhowthisareaisprogressing(6).Thedevelopmentsmayalsotransformthewaythepharmaceuticalindustryworks.EliLilly,forexample,arenowdevelopingmanyoftheirnewtherapiesbasedonspecificbiomarkers(28).

Thenextstepandacurrenthottopicistoprovidefurtherinsightintotheworkingsofthebodyinhealthanddiseasebylookingattheproteinsactiveinparticularcelltypes.ThiscanbeachievedinpartbylookingatthemessengerRNA(transcriptomics)andnoncodingRNAsshowinghowgeneticsaffectsthecellsystemincombinationwithenvironmentalinfluences.Withthespeedatwhichtechnologyisdevelopingperhapseventhesehurdleswillsoonbeovercometoincreasetheefficiencyandefficacyofsequencingandpersonalisedmedicineintheclinic.Thereisnodoubt,however,thattherapidprogresswehaveseensincethefirstsequencingtechnologiesisalreadyhighlyremarkable.

References

(1)http://www.sciencedaily.com/releases/2012/02/120228123847.htm

(2)http://www.bbc.co.uk/news/scienceenvironment17191398

(3)www.nature.com/news/microbiomesequencingoffershopefordiagnostics1.10299)

(4)http://www.sanger.ac.uk/genetics/CGP/

(5)http://www.sciencenews.org/view/generic/id/38320/title/First_complete_cancer_genome_sequenced

(6)Curtisetal(2012),Thegenomicandtranscriptomicarchitectureof2,000breasttumoursrevealsnovelsubgroups,Nature,publishedonlineApril2012,doi:10.1038/nature10983

(7)http://news.sciencemag.org/scienceinsider/2012/05/worldslargesthubforcancer.html?ref=hp

(8)http://blogs.nature.com/news/2012/05/uscancergenomerepositoryhopestospeedresearch.html

(9)Pressrelease:http://www.nanoporetech.com/news/pressreleases/view/39

(10)http://www.nature.com/news/norwaytobringcancergeneteststotheclinic1.9949

(11)HutchinsonIII,C.A.(2007)DNAsequencing:BenchtoBedsideandBeyond.NucleicAcidsResearch,35:18(62276237)doi.10.1093/nar/gkm688

(12)http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1980/

http://obrreview.com/2012/role-of-non-coding-mirna-in-infectious-diseases

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(13)http://www.wellcome.ac.uk/Educationresources/Teachingandeducation/Animations/DNA/WTX056046.htm

(14)http://wellcome.ac.uk/Educationresources/Teachingandeducation/Animations/DNA/WTX056051.htm

(15)Youtubevideo:http://www.youtube.com/watch?v=Sx6FbYoFGmM

(16)http://www.nanoporetech.com/technology/introductiontonanoporesensing/introductiontonanoporesensing

(17)http://www.nature.com/news/nanoporegenomesequencermakesitsdebut1.10051

(18)www.nature.com/news/dnadonorrightsaffirmed1.10275

(19)http://news.sciencemag.org/scienceinsider/2012/03/biobanksaskedtohelpdeliver.html?ref=hp

(20)Robertson,(2003)The$1000Genome:EthicalandLegalIssuesinWholeGenomeSequencingofIndividuals.TheAmericanJournalofBioethics,InFocus.

(21)RebeccaSkloot(2010),TheImmortalLifeofHenriettaLacks,CrownBooks,February2,20101stEdition,ISBN9781400052172

(22)Roche(2010),TheProperty/PrivacyConundrumoverHumanTissue,HECForum22:197209DOI10.1007/s1073001091372

(23)Coryell(2011)PatentLawasanIncentivetoInnovatenotDonate:TheRoleoftheUSPatentSysteminregulatingOwnershipofHumanTissue,PhDThesis?

(24)http://www.sciencemag.org/content/305/5688/1226.2.full.pdf

(25)http://content.healthaffairs.org/content/22/5/166.full

(26)http://www.science20.com/news_articles/nextgeneration_sequencing_leads_personalized_medicine_win_teenager80079

(27)http://www.nature.com/news/2011/110615/full/news.2011.368.html

(28)http://www.pharmamanufacturing.com/articles/2008/007.html

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