GenomeProjects

ShifraBen‐Dor

Bioinforma5csandBiologicalCompu5ngUnit,WeizmannIns5tuteofScience

Outline

  Whataregenomes,andhowweretheysequenced?

  Whatcanwedowithgenomes?

  Howdowelookatgenomes?

  Howdowechooseabrowser?

  Howdothebrowserswork?

WhyStudyGenomes?

  Understandbiologicalprocesses

  Understandpathologicalprocesses

  Diagnose,prevent,andcurediseases

Sowhatisagenome?

 Agenomeisthefullcollec5onofgene5cmaterial(DNA)ofanorganism(includingnon‐nuclearDNA,suchasmitochondrialorchloroplastDNA)

  Itismorethanthegenes(whichareonly~1.5%ofthehumangenome)

  Inhumansthereare3,000,000,000basepairsofDNA

WhatelseisintheDNA?(asidefromgenes)

  Thereareareasthatdon’tcodeforgenesatall

  Thereareareasthatregulatethegenes

–  Whenshouldaproteinbeexpressed?night/day,fetus/adult

–  Whereshouldaproteinbeexpressed?eye,lung,muscle,brain

AGGTTAGATTATGCCCCGAGGGCGCCCCAGCCGAAATTTTTAATGCAGGTTTAATAGTTTAGAGCCTGTGGGCTTCCATGGCTTGGTTCTGCTGTTCTTCACTGGGGACTTGGGGGACCCTGGGAGCTTGTGATGGGGCCTGTCTCCACCTCTGTAAATCCAAGGAGTCAGATGACAAATCTGTCATTTCGGGCGACACACTCCCCTGAGGAAAGGGCCTTGCAGGAGGGCAGAGCAGCTTGCTGGGCATGGCAGGGAGTGGAGAAGGGCAGGGGGCGCAGAGCAGGAGCAGCTTCCTGCCTCTGGGTGGGGACAGTGATCCCCACTGGGGACTGGCAAAGCCCCATGCTCTCTGTTCACCCTGGATGGGTGGCACCTGGGGGCAGGCATGGGGCCTGCAGGAGCCCCTGTGTGCCAGCCCTCCCCTGCCAGCATCCCATCTCCCAGGAGGCCCCCAGGGCAGGTAAGTGCCAGGTCCCCCCTCAGCTCACCGTTGTCCTTCCCCTTGACGAACGCCTCCCACTCCCGGAACCACTGCATGCTGATGCAGTAGATGACGCCCGGCGACTCCTCGGCCTGGAAGGCCTTGTTCAACTGGCAGGCGGGCAGGGACGGGAGGAGACAGAGGGCAGGTCAGTCTCTATTTACCTTCAGCAAGGATTTCCCAAATGCCCCCGCCCCAGTCCCTCACCCAAGAGTCTTACAAAAACACCAGATACTCAAGGTGAAAAATCAAACCCCCAGACAAGCTCAAACAAAATGAACACACCCATCACTCAGAGAGGACGGTGGTAACATTTTGTGGGTCTCTTCAATAACTGTTTGACCCAACTGAGACCACAAGGGAGATTCTACTTTTTGAGAAGGAATCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCGCGATCT CGGCTTACTGCAACCTCTGCCTCCCAGGTTCAAGCGATTCTCCCACCTCAGCCTCTTGAGTAGCTGGGATTACAGGTGTGTGCCACCACCTGGCTACTTTTTGTGTTTTTAGTAGAGACGGGGTTTCGCCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCGCCCACCTCGACCTCTCAAAAGTGCTGGGATAACAGGCATGAACCACTGCGCCCGGCCTGGGAGATGCTAATTTTCTCCGGTTGAATAGAATGTGCCTATCTGCTCAGAGAGGCAGCTCTCCTTCTGACAGGAGCATTTTCTTTTTCGAGATGGGGGGTGGTCTCACTCTGTGCCCAGGCGGGAGTGCAGCGGCGCAATCATGGCTCACTGCAGCCTTGACCTCCTGGGCTCAAGTGATCCTCCCACCTCAGCCTCCTGAGTAGGTTGGACCACAGGTGCATACCACTAGGCCCAGCCCTGACAGTCTCTTTTTCGTTTGTGTTCTGAGACAGGGTCTCACTCTATTGCCCAGGCTGCGGTGCAGTGGCATGATCACGGCTCACTGCAGCCTCAACCTCCCAGGCTTAGGTGATCCTCCCAACTCACTCAGCCCTCCAGGTAGCGGGGACTACAGGTACACATCACCATGCCTGGCTAATTTTTGTATTGTTTGTAGAGATGGGGTTTCGCCATGTTGGCCAAGTTGGTCTTGAACTCCTGG

ChallengesofDNA

  DNAhasonlyfourleZers

  TheyarestrungtogetherwithNOobviouspunctua5on

  Therearenosignalstosay“agenestarts(orends)here”

  Howdowemakesenseofsomuchinforma5on?

Ifcompiledinbooks,thedatawouldfillan

es5mated200volumesthesizeofa

ManhaZantelephonebook(at1000pages

each),andreadingitwouldrequire26

yearsworkingaroundtheclock.

TheHumanGenomeProject

  Wasplannedin1988,startedin1990

  Originallyplannedtotake15years,inthreefiveyearstages

Objec5onstothegenomeproject

1)Fearthatfundingwillbedivertedfromotherareasofresearch

2)Whatisthevalueofsequencingacompletegenome,giventhehighpropor5onofnongenicsequences(“JunkDNA”).

Twoaltera5onsintheoriginalplanhelped:

1)Focusshifedfromlarge‐scalesequencingtomappingthegenome,whichwouldhastenthesearchfordiseasegenes

2)Simultaneouslydeterminethenucleo5desequenceofthegenomesofotherorganisms;thisprovidescomparisonsandpointsofreferenceforthehumansequence

Stage11991‐1995

  Crea5ngaGene5cMapofthegenome

  Crea5ngaPhysicalMapofthegenome

  Crea5ngasetofoverlappingclones

  Createfaster/cheapermethodsofsequencing

  Createsofware/databasesthatcandealwiththedata

Stage21994‐1998

  Finishmapping(bothgene5candphysical)

  Startsequencing

  Startannota5on‐genefindingandplacementonmaps

Stage 3 1998-2003 Area Goals 1993-98 Status as of Oct. 1998 Goals 1998-2003 Genetic map Average 2- 1 cm map published Completed to 5-cm Sept. 1994

Physical map Map 30,000 STSs 52,000 STSs mapped Completed

DNA sequence Complete 80 Mb 180 Mb human plus Finish 1/3 of human for all organisms 111 Mb non-human sequence by end of 2001 by 1998 Working draft of

remainder end of 2001 Complete human sequence by end of 2003

Human sequence Not a goal - 100,000 mapped SNPs variation

Gene Develop 30,000 ESTs mapped Full-length cDNAs Identification Technology

Functional Not a goal - Develop genomic-scale analysis technologies

Techniques

ThemajorbreakthroughthatallowedtheHumanGenomeProjecttotakeoffweretwotechniques:

1)  Improvementsinsequencing:CycleSequencingAutomatedSequencersFlourescentDyesHighThroughput‐lowercosts

2)PCR

Markers

Therearemanydifferenttypesofmarkers:

RFLP:Restric5onFragmentLengthPolymorphism

Microsatellite

VNTR:VariableNumberTandemRepeat

STS:SequenceTaggedSite

1 2

100 200

400

500

Restric5onFragmentLengthPolymorphism

700basepairPCRfragment

Allele1

Allele2

GAATTC

GAATTC

GAATTC

GAATTT

Microsatellites

Microsatellitesaresmallrepe55vestretches

ofDNA,usuallyrepeatsofdi,triandtetra

nucleo5des.

ForexampleCACACA,orCAGCAGCAG…..

Becausethesestretchesrepeat,whenthe

DNArecombines,itsveryeasyforthe

machineryto“slip”andaddorsubtractafew

copies

1 2

4 2

Allele

# of repeats

AAAACAGCAGCAGTTTTT AAAACAGCAGCAGTTTTT

AAAACAGCAGCAGTTTTT AAAACAGCAGCAGTTTTT

Parent chromosomes Recombination

AAAACAGCAGCAGCAGTTTTT

AAAACAGCAGTTTTT

Daughter chromosomes Allele 1

Allele 2

VariableNumberTandemRepeat

RegionsofDNAthatrepeat‐forexample,microsatellites,althoughVNTR’scanhavemorecomplexsequence.

ThisisalsodetectedbyperformingPCRandlookingatthenumberofrepeatsonagel

primer 1

Primer 2

PCR product

A genomic region

Sequence Tagged Site

STS is defined as:

primer 1 primer 2

PCR product size STS is designed to be unique in the genome

Maps

Therearetwotypesofmaps:

Gene5c‐measuresrecombina5ondistance

Physical‐measuresphysicaldistance

Gene5cMapsAgene5cmapmeasuresrecombina5ondistanceandanswerstheques5on,“Howofenaretwomarkersfoundtogether?”

Twomarkersaresaidtobe1cen5Morgan(cM)apartiftheyareseparatedbyrecombina5on1%ofthe5me.Agene5cdistanceof1cMisroughlyequaltoaphysicaldistanceof1millionbasepairs(1MegabaseorMb)

Gene5cMaps

5 2 4 4

2 4 2 4

2 3 1 4

3 2 2 4

2 4 2 4

2 5 1 4

2 4 2 4

2 3 1 4

2 4 2 4

2 5 2 4

2 4 2 4

2 3 1 4

2 4 2 4

2 5 2 4

3 2 2 4

2 5 2 4

3 2 2 4

2 5 2 4

PhysicalMaps

Physicalmapsvarygreatly.Thelowestresolu5onmaparethechromosomebandingpaZerns(ideogram).

Thehighestresolu5onmapistheactualsequence.

Whatweactuallyuseisinbetween.Onetypeofmapdevelopedaspartofthegenomeprojectistheradia5onhybridmap(RHmap)

3 12 82 83

STS C

STS B

Chromosome

STS X

STS Y

STS Z

Firstrarecuongenzymeswereused

togeneratepieces~150,000basepairs

long.Differentenzymeswereusedto

getoverlappingsegments.

Howwasthegenomecloned?

Marker

Enzyme site

BACs(BacterialAr5ficialChromosomes)wereplacedalongthemapwiththehelpofmarkers,andtheleastredundantpathwaschosen.

TheneachBACwasbrokendownthesameway,thefragmentswereplacedinorderusingrestric5onenzymemapping,andsequenced.

p q Digest

Define a set of overlapping clones

Sequence

CeleraCorp.saidthatitwouldsequencethegenomefasterandcheaperthanthepublicprojectcould.

Theyusedamethodknownasrandomshotgunsequencing.

Theybrokethegenomeupinto2000and10,000bppieces,sequencedthem,andwroteacomputerprogramtoputitallbacktogether.

ThencameCelera…..

p q Shotgun digest

Sequence

Take random clones

WholeGenomeShotgun

Wholegenomeshotgundidn’tactuallysequencewholepiecesofDNA,butjusttheirends.

TheWGSreadsaverage~500bp.

Theydohowevergiveinforma5onintermsofmatepairsastowhichpiecefallswhere,andthatgivesamethodoforderingandameasureofthesizeoftheholes

Mate Pair

Contigs

NNNNN NN NNNN

Scaffold or Supercontig

Itisimportanttonotethatnotallgenome

sequencesareorganizedintochromosomes.

Thosethatarebeingsequencedbywhole

genomeshotgun,wheremappinghasnot

takenplace,areorganizedinto“linkage

groups”

Theproblemswithshotgun…

Thereareseveralproblemswithshotgunsequencing:

‐ repe55veelements

‐ genefamilies

‐needmoresequence

“x”coverage

 Whatdoes5xcoverageofthegenomemean?

 That55mesthenumberofbasesinthatgenomeweresequenced(soforhuman5x3x109bases)

  ItdoesNOTmeanthatthewholegenomewascovered55mes

Thepublicreac5on….

ThepublicprojectwasconcernedthatCelerawouldfinishfirst,andasacommercialcompany,trytopatentsignificantpartsofthegenome.

Tomakethepubliceffortfaster,theydecidedtoshotguntheexis5ngBACs.

ThisleadtoHTGs

HighThroughputGenomesequence

Theoutputofthepublicprojecthadbeenwellorderedwellfinishedsequence.

Asaresultofshotgunsequencing,weendedupwith“draf”sequence‐sequencewhosegeneralloca5onisknown,buttheexactorderordirec5onofthepiecesisnot.

HTGS

Status Loca-on Defini-on

Phase0 HTGdivision single‐fewpassreadsofa singleclone(notcon5gs).

Phase1 HTGdivision Unfinished,maybe unordered,unorientedcon5gs, withgaps.

Phase2 HTGdivision Unfinished,ordered,oriented con5gs,withorwithoutgaps.

Phase3 Primarydivision Finished,nogaps(withor withoutannota5ons).

Dra4Sequence‐Spring2000p q

GapOrderedcon5gs,

finishedandunfinished

Orienta5onUnknown

?

?

UnfinishedClone

?

?

UnmappedCon5gs

p q

mRNA

Genomic DNA

?

?

1 4500

20,450 125,000 Draft sequence

SequencingProblems

  PhysicalHoles(noclones)  SequenceHoles(nosequence)

AssemblyProblems

  Repe55veelements  Genefamilies

  Pseudogenes

  Duplica5on

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Thepost‐genomicera…..

Nowthatwehavemostofthegenomesequence,effortsarebeingturnedtounderstandingthewealthofinforma5onproduced:

Definingthegenes,whenandwheretheyareexpressed,whatcontrolsthem,whotheyinteractwith,andhowtheyaremutated,par5cularlyindisease.

AdvantagesofGenomeSequence

  Previously,itwas“onegene,onepostdoc”

  NowthatwehaveabeZerpictureofthings,wecanstudysystems,andgeneinterac5ons

  Previously,ittookyearstocloneandsequenceagene

  Now,allweneedisaliZlebitofsequence,

andwecanlookuptherestinthegenome

AdvantagesofGenomeSequence

 Previously,aferyearsspentcloning,more5mewasneededtosequencethesurroundingareaofthegene,tostartlookingintoregulatoryelements

 Now,wehavethesurroundingsequenceandcanstartlookingfortheregulatoryelementsdirectly

Sowestartedwithgenomics….

Nowwehave“Omics”:

  Transcriptome

  Proteome

  Regulome…….

Ands5llplentyofwork…….

StatesofCommonGenomes

http://www.genomesonline.org

NoEukaryo5cGenomeisFinished

  Euchroma5n/Heterochroma5n

  Duplica5ons

  Gaps

N50

  Acommonmeasureforhowwellput

togetheragenomeis

  Themarkwhere50%oftheclonesare

aboveorbelowthatlength

  N50human:46.4Mb(build37)

  N50mouse:39.3Mb(build37)

HumanGenome‐Build37

Human Build 37

“TheYchromosomeinthisassemblycontainstwopseudoautosomalregions(PARs)thatweretakenfromthecorrespondingregionsintheXchromosomeandareexactduplicates:

chrY:10001-2649520 and chrY:59034050-59363566 chrX:60001-2699520 and chrX:154931044-155260560

UCSCgenomebrowser,humangenomepage

YChromosome

MouseGenome‐Build37

MouseGenome

NCBIMouseBuild36representsahighlypolishedproduct.Itiscomposedlargelyoffinishedsequence(clickhereforagraphofsequencecomposi5on).HTGSphase3sequenceisassembledbyhandintonon‐redundantcon5gsandthenon‐redundantsequenceisusedasanimputtotheassembly.AhandcuratedcombinedTilingPathfilewasusedtoguidetheassembly.Allnon‐finishedpor5onsoftheassemblyhavebeenhandcuratedtodeterminewhethertoincludeWGS,HTGSsequenceortoleaveagap.

Finished Draft WGS Non-HTG Seq Gap

Mouse Build 37

Mousethingstoremember

 Strains– ThereferencesequenceisfromC57Bl/6J

– Thereissequenceavailablefrom:129,A/J,B6/CBAF1J,Balb/c,C3H,NOD

 Ychromosome‐theoriginalreferencemousewasfemale!

MouseGenome

TheYchromosomewasbuiltbyhandincollabora5onwiththeWashingtonUniversityGenomeSequencingCenterandthePagelabattheWhiteheadIns5tuteforBiomedicalResearch.Currently,onlytheshortarmoftheYhasreliablemappingdata,somostofthecon5gsontheYchromosomeareunplaced.

Rat Genome

“final”versioninbrowsers:September/October2003

Officialrelease:updatedin2008

Release 4:

Release5:2006

Table1:Release5Sta5sicsArm Gaps MajordifferencecomparedtoRelease4X 3 8kbaddedtothedistalend, gapsfilledinregions1‐112L 2 591kbaddedtotheproximalendofthearm.2R 1 380kbaddedtotheproximalend.3L 1 16kbaddedondistalend,

718kbaddedtoproximalend,othergapsfilled.3R 0 None4  1 70kbpaddedtothedistalend

Gapsofunknownsizearedenotedby100N'sinthefastafiles.TherearetwosizedgapsonXthathavees5matesfortheirsize.Thereare6othergapsinthegenomewhicharenotsized.Inaddi5ontothemajorchanges,allarmsexceptfor3Rhadminorsequencechangesinregionswherethefingerprintdigestshowedevidenceofmissassembly,orinregionsthatrequiredqualityimprovements.

Release5:donein2006

Arabidopsis–TAIR10

Thesequencedregionscover119.1megabasesofthe125‐megabasegenomeandextendintocentromericregions.Majorreleasein2003,minorupdatesin2008,2009.Hasalistofknowngapsinthegenome.

TheTAIR10releasecontains27,416proteincodinggenes,4827pseudogenesortransposableelementgenesand1359ncRNAs(33,602genesinall,41,671genemodels).Atotalof126newlociand2099newgenemodelswereadded.

TAIRGenomeMonitoringSeptember6,2002

WeregularlycomparemRNAandESTsequencesfromGenBankandTIGRwiththeArabidopsisgenomesequenceandpulloutArabidopsissequencesforwhichthereisnogoodmatch(eitherbecausethesequencequalityistoolow,theyarefromanotherorganism,ortheyfallintooneofthefewsmallunsequencedregionsremaining).GotoourFTPsitetodownloadfastafilescontainingtheseunmatchedsequences.

TotalmRNAstested:17,441Numberthatdidnotmatchthegenomesequence:19(0.0011%).TotalESTstested:174,625Numberthatdidnotmatchthegenomesequence:1600(0.009%).

Ideasandslidestakenfrom:

IritOrrVeredChalifaCaspiDolanDNALearningCenterHumanGenomeProjectatDOEYourGenome‐SangerCenter