10/29/2015

What'saGenome?

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What'saGenome?

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HowdoesDNAsequencingwork?

What'saChromosome?
What'saGene?
What'sDNA?

Regardlessoftheapproachtothegenomeasawhole,theactualprocessofDNAsequencing
isthesame.Sequencingemploysatechniqueknownaselectrophoresistoseparatepiecesof
DNAthatdifferinlengthbyonlyonebase.

Inelectrophoresis,DNAtobesequencedisplacedatone
What'sGenomeSequencing? endofagelaslabofagelatinlikesubstance.(Amajor
partofDNAsequencingsimplycomesdowntomaking
Whatisgenome
abunchofJellO.)Electrodesareplacedateitherendof
sequencing?
thegelandanelectricalcurrentisapplied,causingthe
Whyisgenome
DNAmoleculestomovethroughthegel.Smaller
sequencingso
moleculesmovethroughthegelmorerapidly,sothe
important?
DNAmoleculesbecomeseparatedintodifferentbands
Howdoyousequencea
accordingtotheirsize.Thecatchisthatelectrophoresis
genome?
canonlyseparateabout500basesintoclearbands
HowdoesDNA
hencetheneedforchoppingDNAupintosmallpieces
sequencingwork?
inordertosequenceit.
Howdoesthe
sequencingmachine
knowwhetherabaseis
anA,C,G,orT?
WhathappensafterDNA
sequencescomeoutof
thesequencing
machines?
Howdoyouassemblea
genome?
Howdoscientistsknowif
agenomesequenceis
right?
Whatmakessequencing
thehumangenome
differentfromsequencing
othergenomes?
Whenisagenome
sequencedone?

What'sagenomemap?
Whataregenomevariations?
GNNHome

Untilthelate1980s,electrophoresisgelswerealways
readbyaperson.EachpieceofDNAwasattachedtoa
radioactivelabel,andanXraypicturewasmadeofthe
geltomakethepositionsoftheDNAbandsvisible.
Labwithsequencingmachines
Painstakinglyanalyzingtherowsandcolumnsofbands CourtesyofCeleraGenomics
onthegel,apersoncoulddeterminethesequenceoftheDNA.
Butthisprocesswasslow,tedious,andfraughtwitherror.Today'slargescalesequencing
projectswouldbeimpossiblewithoutautomaticsequencingmachines,whichbecame
commerciallyavailableinthelate1980sandhavemadeDNAsequencingmuchquickerand
morereliable.Inoneyear,apersoncanproduceafinishedsequenceof20,000to50,000
basesamachinecanproducearoughdraftofasequencethatlonginjustafewhours.
Mostautomaticsequencingmachineshaveadesignbasedcloselyontheoriginal,manual
sequencingprocess.Torunthemachine,atechnicianpoursgelintothespacebetweentwo
glassplatessetlessthanhalfamillimeter(twohundredthsofaninch)apart.Afterthegel
sets,DNAisloadedintoeachofthe96lanesjustlikethelanesonahighwayorinapool
thatrunthelengthofthe30cm(about1foot)gel.AstheDNApiecesmovethroughthe
gel,thesequencingmachinereadstheorderofDNAbasesandstoresthisinformationinits
computermemory.
Insomenewermachines,knownascapillarysequencers,DNAisrunthroughanarrayof96
gelfilledcapillariesglasstubesaboutthewidthofahumanhairratherthanthrougha
slabofgel.Butjustliketheslabgelmachines,capillarymachinesreadthebasesequenceas
DNAmovesthroughthegel.

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What'saGenome?

Closeupofcapillariesfromacapillarysequencingmachine
CourtesyofCeleraGenomics

CapillarysequencerscansequenceeachpieceofDNAabouttwiceasfastasslabgel
machines.Moreover,theyarefullyautomatedaroboticarmplacestheDNAintothetop
ofthecapillaries.Themachineautomaticallyfillsthecapillarieswithgelandcleansthem
betweenruns,soonlyaminimumofhumanattentionabout15minutesadayis
necessarytorefillthecontainersofgel,water,andothersolutionslocatedinthemachine's
"guts."Ontheotherhand,sequencingmachinesareexpensiveandcapillarysequencersare
sonewthatsomelabshavehadtroublegettingthemtoworkattopefficiency.Mostlarge
scalesequencingprojectsuseacombinationofslabgelandcapillarymachines.

Howdoesthesequencingmachineknow
whetherabaseisanA,C,G,orT?
Sequencingmachinescan't"see"DNAdirectly,soscientistsmustuseacomplexsetof
procedurestoprepareDNAforsequencing.WhenDNAisfinallyinaformthatthe
machinescanread,ithasbeenchoppedup,copied,chemicallymodified,andtaggedwith
fluorescentdyescorrespondingtothefourdifferentDNAbases,orgeneticletters.
Beforeitissequenced,apieceofDNAiscopiedmanytimes,thendividedintofourbatches
inpreparationforanotherroundofcopying.Inthissecondround,asmallamountof
chemicallymodifiedbaseisaddedtoeachbatchthatis,modifiedTtoonebatch,Ato
another,andsoon.WhenoneofthesemodifiedbasesisincorporatedintoaDNAmolecule,
thechainofbasesstopsgrowing.TheresultofallthisisthatonebatchofDNAwillcontain
onlypiecesthatendinT,anotheronlypiecesthatendinA,athirdonlypiecesthatendin
G,andthefourthbatchonlypiecesthatendinC.

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Inthesecondroundofcopying,adifferentfluorescentdyeisalsoaddedtoeachbatchof
DNA.Thus,everypieceofDNAthatendswithThasabluedyetag,forexamplethose
thatendinAhaveareddyetagthosethatendinGhaveayellowdyetagandthosethat
endinChaveagreendyetag.
SupposeyouapplythatproceduretothissequenceofDNA:
TAGACT
Attheendofthesecondroundofcopying,eachbatchwillcontainthefollowingpiecesof
DNA:
1:blueT,blueTAGACT
2:redTA,redTAGA
3:yellowTAG
4:greenTAGAC
IntoonelaneorcapillaryofasequencingmachinegoesamixtureofDNAfromallfour
batches.Becausesmallermoleculesmovethroughthegelfaster,theDNApiecescome
throughthegelinincreasingorderofsizeeachpieceonebaselongerthanthelast.
Thus,inthisexample,thefirstpiecetomakeitallthewaythroughthegelisaTattached
toabluedyetagthenextpieceisTAwithareddyetagnextisTAGattachedtoayellow
dyetagandsoon.

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Asthepiecesemergefromthegel,theymovepastalaserthatcausesthedyemoleculesto
fluoresce.Adetectorreadsthecolorofthefluorescenceblue,red,yellowanda
softwareprogrammatchesthecolortothecorrespondingbaseT,A,G.Inthisway,the
sequencegrowsbasebybase.Eachsequenceof500basesorsothatasequencingmachine
generatesisknownasa"read."

WhathappensafterDNAsequencescome
outofthesequencingmachines?
Anautomaticsequencingmachinespitsoutwhatgenomescientistscall"raw"sequence.In
rawsequence,thereadsorshortDNAsequencesarealljumbledtogether,likethepiecesof
ajigsawpuzzleinajustopenedbox.Inevitably,rawsequencealsocontainsafewgaps,
mistakes,andambiguities.
Theprocessofpolishingthatrawsequencetransformingthefragmentedroughdraftintoa
long,continuousfinalproductwithoutbreaksorerrorsiscalledfinishing.Finishing
involvesbothassembly,inwhichindividualreadsarehookedtogetherintheproperorder,
andalaboriousprocessofdoublecheckingandrefiningthesequencetoeliminatemistakes
andclosegaps.Finishingoftentakeslongerthanthesequencingitself.
.....................
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