Towards complete and error-free genome assemblies of all

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Advertisement nature articles article Towardscompleteanderror-freegenomeassembliesofallvertebratespecies DownloadPDF Subjects EvolutionarygeneticsGenomeassemblyalgorithmsMolecularevolutionResearchdata AbstractHigh-qualityandcompletereferencegenomeassembliesarefundamentalfortheapplicationofgenomicstobiology,disease,andbiodiversityconservation.However,suchassembliesareavailableforonlyafewnon-microbialspecies1,2,3,4.Toaddressthisissue,theinternationalGenome10K(G10K)consortium5,6hasworkedoverafive-yearperiodtoevaluateanddevelopcost-effectivemethodsforassemblinghighlyaccurateandnearlycompletereferencegenomes.Herewepresentlessonslearnedfromgeneratingassembliesfor16speciesthatrepresentsixmajorvertebratelineages.Weconfirmthatlong-readsequencingtechnologiesareessentialformaximizinggenomequality,andthatunresolvedcomplexrepeatsandhaplotypeheterozygosityaremajorsourcesofassemblyerrorwhennothandledcorrectly.Ourassembliescorrectsubstantialerrors,addmissingsequenceinsomeofthebesthistoricalreferencegenomes,andrevealbiologicaldiscoveries.Theseincludetheidentificationofmanyfalsegeneduplications,increasesingenesizes,chromosomerearrangementsthatarespecifictolineages,arepeatedindependentchromosomebreakpointinbatgenomes,andacanonicalGC-richpatterninprotein-codinggenesandtheirregulatoryregions.Adoptingtheselessons,wehaveembarkedontheVertebrateGenomesProject(VGP),aninternationalefforttogeneratehigh-quality,completereferencegenomesforalloftheroughly70,000extantvertebratespeciesandtohelptoenableaneweraofdiscoveryacrossthelifesciences. 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MainChromosome-levelreferencegenomesunderpinthestudyoffunctional,comparative,andpopulationgenomicswithinandacrossspecies.Thefirsthigh-qualitygenomeassembliesofhuman1andothermodelspecies(forexample,Caenorhabditiselegans2,mouse3,andzebrafish4)wereputtogetherusing500–1,000-basepair(bp)Sangersequencingreadsofthousandsofhierarchicallyorganizedcloneswith200–300-kilobase(kb)inserts,andchromosomegeneticmaps.Thisapproachrequiredtremendousmanualeffort,softwareengineering,andcost,indecade-longprojects.Whole-genomeshotgunapproachessimplifiedthelogistics(forexample,inhuman7andDrosophila8),andlaternext-generationsequencingwithshorter(30–150-bp)sequencingreadsandshortinsertsizes(forexample,1 kb)usheredinmoreaffordableandscalablegenomesequencing9.However,theshorterreadsresultedinlower-qualityassemblies,fragmentedintothousandsofpieces,wheremanygenesweremissing,truncated,orincorrectlyassembled,resultinginannotationandothererrors10.Sucherrorscanrequiremonthsofmanualefforttocorrectindividualgenesandyearstocorrectanentireassembly.Genomicheterozygosityposedadditionalproblems,becausehomologoushaplotypesinadiploidorpolyploidgenomeareforcedtogetherintoasingleconsensusbystandardassemblers,sometimescreatingfalsegeneduplications11,12,13,14.Toaddresstheseproblems,theG10Kconsortium5,6initiatedtheVertebrateGenomesProject(VGP;https://vertebrategenomesproject.org)withtheultimateaimofproducingatleastonehigh-quality,nearerror-freeandgapless,chromosome-level,haplotype-phased,andannotatedreferencegenomeassemblyforeachofthe71,657extantnamedvertebratespeciesandusingthesegenomestoaddressfundamentalquestionsinbiology,disease,andbiodiversityconservation.Towardsthisend,havinglearnedthelessonsofhavingtoomanyvariables thatmakeconclusionsmoredifficulttoreachintheG10KfromtheG10KAssemblathon2effort15,wefirstevaluatedmultiplegenomesequencingandassemblyapproachesextensivelyononespecies,theAnna’shummingbird(Calypteanna).Wethendeployedthebest-performingmethodacrosssixteenspeciesrepresentingsixmajorvertebrateclasses,withawidediversityofgenomiccharacteristics.Drawingontheprincipleslearned,weimprovedthesemethodsfurther,discoveredparametersandapproachesthatworkbetterforspecieswithdifferentgenomiccharacteristics,andmadebiologicaldiscoveriesthathadnotbeenpossiblewiththepreviousassemblies.Complete,accurateassembliesrequirelongreadsWechoseafemaleAnna’shummingbirdbecauseithasarelativelysmallgenome(about1 Gb),isheterogametic(hasbothZandWsexchromosomes),andhasanannotatedreferenceofthesameindividualbuiltfromshortreads16.Weobtained12newsequencingdatatypes,includingbothshortandlongreads(80 bpto100 kb),andlong-rangelinkinginformation(40 kbtomorethan 100Mb),generatedusingeighttechnologies(SupplementaryTable1).Webenchmarkedalltechnologiesandassemblyalgorithms(SupplementaryTable2)inisolationandinmanycombinations(SupplementaryTable3).Toourknowledge,thiswasthefirstsystematicanalysisofmanysequencetechnologies,assemblyalgorithms,andassemblyparametersappliedonthesameindividual.Wefoundthatprimarycontiguoussequences(contigs)(pseudo-haplotype;SupplementaryNote 1)assembledfromPacificBiosciencescontinuouslongreads(CLR)orOxfordNanoporelongreads(ONT)wereapproximately30-to300-foldlongerthanthoseassembledfromIlluminashortreads(SR),regardlessofdatatypecombinationorassemblyalgorithmused(Fig.1a,SupplementaryTable3).ThehighestcontigNG50sforshort-read-onlyassemblieswereabout0.025to0.169 Mb,whereasforlongreadstheywereabout4.6to7.66 Mb(Fig.1a);contigNG50isanassemblymetricbasedonaweightedmedianofthelengthsofitsgaplesssequencesrelativetotheestimatedgenomesize.AfterfixingafunctioninthePacBioFALCONsoftware17thatcausedartificialbreaksincontigsbetweenstretchesofhighlyhomozygousandheterozygoushaplotypesequences(SupplementaryNote 1,SupplementaryTable2),contigNG50nearlytripledto12.77 Mb(Fig.1a).Thesefindingsareconsistentwiththeoreticalpredictions18anddemonstratethat,givencurrentsequencingtechnologyandassemblyalgorithms,itisnotpossibletoachievehighcontigcontinuitywithshortreadsalone,asitistypicallyimpossibletobridgethroughrepeatsthatarelongerthanthereadlength.Fig.1:ComparativeanalysesofAnna’shummingbirdgenomeassemblieswithvariousdatatypes.a,ContigNG50valuesoftheprimarypseudo-haplotype.b,ScaffoldNG50values.c,Numberofjoins(gaps).d,Numberofmis-joinerrorscomparedwiththecuratedassembly.Thecuratedassemblyhasnoremainingconflictswiththerawdataandthusnoknownmis-joins.*SameasCLR + linked + Opt. + Hi-C,butwithcontigsgeneratedwithanupdatedFALCON17versionandearlierHi-CSalsaversion(v2.0versusv2.2;SupplementaryTable2)forlessaggressivecontigjoining.e,f,Hi-Cinteractionheat mapsbeforeandaftermanualcuration,whichidentified34chromosomes.Gridlinesindicatescaffoldboundaries.Redarrow,examplemis-jointhatwascorrectedduringcuration.g,Karyotypeoftheidentifiedchromosomes(n = 36 + ZW),consistentwithpreviousfindings70.h,Correlationbetweenestimatedchromosomesizes(inMb)basedonkaryotypeimagesingandassembledscaffoldsinSupplementaryTable4(bCalAna1)onalog–logscale.v1.0,VGPassemblyv1.0pipeline;linked,10XGenomicslinkedreads;Hi-C,Hi-Cproximityligation;1D,2D,OxfordNanoporelongreads;NRGene,NRGenepaired-endIlluminareads;SR,paired-endIlluminashortreads.FullsizeimageIterativeassemblypipelineScaffoldsgeneratedwithallthreescaffoldingtechnologies(thatis,10XGenomicslinkedreads(10XG),Bionanoopticalmaps(Opt.),andArimaGenomics,DovetailGenomics,orPhaseGenomicsHi-C)wereapproximately50%to150%longerthanthosegeneratedusingoneortwotechnologies,regardlessofwhetherwestartedwithshort-orlong-read-basedcontigs(Fig.1b,ExtendedDataFig.1a,SupplementaryTable3).Thesefindingsincludeimprovementswemadetoeachapproach(SupplementaryNote 1,SupplementaryTables4,5,SupplementaryFig.1).Despitesimilarscaffoldcontinuity,theshort-read-onlyassemblieshadfromabout18,000toabout70,000gaps,whereasthelong-readassemblieshadsubstantiallyfewer(about400toabout4,000)gaps(Fig.1c).Manygapsintheshort-readassemblieswereinrepeatorGC-richregions.Consideringthecuratedversionofthisassemblytobemoreaccurate,wealsoidentifiedroughly5,000to8,000mis-joinsinshort-read-basedassemblies,whereaslong-read-basedassemblieshadonlyfrom20toaround700mis-joins(Fig.1d).Thesemis-joinsincludedchimericjoinsandinversions.Afterwecuratedthisassemblyforcontamination,assemblyerrors,andHi-C-basedchromosomeassignments(Fig.1e,f),thefinalhummingbirdassemblyhad33scaffoldsthatcloselymatchedthechromosomekaryotypeinnumber(33of36autosomesplussexchromosomes)andestimatedsizes(approximately2to200 Mb;Fig.1g,h),withonly1to30gapsperautosome(bCalAnn1inSupplementaryTable6).Ofthefiveautosomeswithonlyonegapeach,three(chromosomes14,15,and19)hadcompletespanningsupportbyatleasttwotechnologies(reliableblocks,ExtendedDataFig.1c;bCalAnn1inSupplementaryTable6),indicatingthatthechromosomecontigswerenearlycomplete.However,theyweremissinglongarraysofvertebratetelomererepeatswithin1 kboftheirends(ExtendedDataFig.1c;bCalAnn1inSupplementaryTables6,7).AssemblypipelineacrossvertebratediversityUsingtheformulathatgavethehighest-qualityhummingbirdgenome,webuiltaniterativeVGPassemblypipeline(v1.0)withhaplotype-separatedCLRcontigs,followedbyscaffoldingwithlinkedreads,opticalmaps,andHi-C,andthengapfilling,basecallpolishing,andfinallymanualcuration(ExtendedDataFigs.2a,3a).Wesystematicallytestedourpipelineon15additionalspeciesspanningallmajorvertebrateclasses:mammals,birds,non-avianreptiles,amphibians,teleostfishes,andacartilaginousfish(SupplementaryTables8,9,SupplementaryNote 2).Forthezebrafinch,weusedDNAfromthesamemaleaswasusedtogeneratethepreviousreferencegenome19,andincludedafemaletrioforbenchmarkinghaplotypecompleteness,wheresequencedreadsfromtheparentswereusedtobinparentalhaplotypereadsfromtheoffspringbeforeassembly20(ExtendedDataFigs.2a,3b).Wesetinitialminimumassemblymetricgoalsof:1 MbcontigNG50;10 MbscaffoldNG50;assigning90%ofthesequencetochromosomes,structurallyvalidatedbyatleasttwoindependentlinesofevidence;Q40averagebasequality;andhaplotypesassembledascompletelyandcorrectlyaspossible.Whenthesemetricswereachieved,mostgeneswereassembledwithgaplessexonandintronstructures11,andfewerthan3%hadframe-shiftbaseerrorsidentifiedinannotation.Q40isthemathematicalinflectionpointatwhichgenesgofromusuallycontaininganerrortousuallynot21.Ofthecuratedassemblies(SupplementaryTable10,SupplementaryNote 2),16of17achievedthedesiredcontinuitymetrics(ExtendedDataTable1).ScaffoldNG50wassignificantlycorrelatedwithgenomesize(Fig.2a),suggestingthatlargergenomestendtohavelargerchromosomes.Onaverage,98.3%oftheassembledbaseshadreliableblockNG50srangingfrom2.3to40.2 Mb;collapsedrepeatbases22withabnormallyhighCLRreadcoverage(morethan3s.d.)rangedfrom0.7to31.4 MbperGb;andthecompletenessofthegenomeassembliesrangedfrom87.2to98.1%,withlessthan4.9%falselyduplicatedregions,consistentwiththefalseduplicationratewefoundfortheconservedBUSCOvertebrategeneset(ExtendedDataTable1,SupplementaryTables11,12).Fig.2:Impactofrepeatsandheterozygosityonassemblyquality.a,CorrelationbetweenscaffoldNG50andgenomesizeofthecuratedassemblies.b,NonlinearcorrelationbetweencontigNG50andrepeatcontent,beforeandaftercuration.c,CorrelationbetweennumberofgapsperGbassembledandrepeatcontent.d,Correlationbetweenprimaryassemblysizerelativetoestimatedgenomesize(y axis)andgenomeheterozygosity(x axis),beforeandafterpurgingoffalseduplications.Assemblysizesabove100%indicatethepresenceoffalseduplicationsandthosebelow100%indicatecollapsedrepeats.e,f,Correlationsbetweengenomeduplicationrate usingk-mers23(e)andconservedBUSCOvertebrategeneset(f),andgenomeheterozygositybeforeandafterpurgingoffalseduplications.g,h,Asine,f,butwithwhole-genomerepeatcontentbeforeandafterpurgingoffalseduplications.Genomesize,heterozygosity,andrepeatcontentwereestimatedfrom31-mercountsusingGenomeScope71,exceptforthechannelbullblenny,astheestimateswereunreliable(see Methods).Repeatcontentwasmeasuredbymodellingthek-mermultiplicityfromsequencingreads.SequenceduplicationrateswereestimatedwithMerqury23using21-mers.*P 1&&(GT=’’AA’’||GT = ‘’Aa’’)’-Hla.VGPTrioPipelinev1.0–v1.6Thetriopipelineissimilarlydesignedtothestandardpipeline,exceptfortheuseofparentaldata(ExtendedDataFig.3b).Whenparentalgenomesareavailable,thechild’sCLRreadsarebinnedtomaternalandpaternalhaplotypes,andassembledseparatelyashaplotype-specificcontigs(haplotigs)usingTrioCanu20.Inbrief,parentalspecificmarkerk-merswerecollectedusingMeryl23fromtheparentalIlluminaWGSreadsoftheparents.Thesemarkerswerefilteredandusedtobinthechild’sCLRread.Ahaplotypewasassignedgiventhemarkersobserved,normalizedbythetotalmarkersineachhaplotype.Thesubsequentpurging,scaffolding,andpolishingstepsweresimilarlyupdatedwiththeuseofPurge_Dups14(v1.6).WeextendedbinningtolinkedreadsandHi-Creads,byexcludingreadpairsthathadanyparental-specificmarker.ThebinnedHi-Creadswereusedtoscaffolditshaplotypeassembly,andpolishedwiththebinnedlinkedreadsfromtheobservationofhaplotypeswitchingusingthestandardpolishingapproach.Duringcuration,oneofthehaplotypeassemblieswiththehigherQVand/orcontiguitywaschosenastherepresentativehaplotype.Theheterogameticsexchromosomefromtheunchosenhaplotypewasaddedtotherepresentativeassembly.However,whilecuratingseveraltrios,wefoundthatinregionsoflowdivergencebetweensharedparentalhomogameticsexchromosomes(thatis,XorZ),asmallfractionofoffspringCLRdatawasmis-assignedtothewronghaplotype.Thismis-alignmentresultedinaduplicate,low-coverageoffspringXorZassemblyinthepaternal(formammals)ormaternal(forbirds)haplotype,respectively,whichrequiredremovalduringcuration.Weareworkingonmethodstoimprovethebinningaccuracyforresolutionofthisissuegoingforward.Forthefemalezebrafinchinparticular,contigsweregeneratedbeforethebinningwasautomatedintheCanuassemblerasTrioCanu1.7,andthereforeamanualbinningprocesswasappliedasdescribedintheoriginalTrio-binningpaper20(SupplementaryMethods).Contigswereassembledforeachhaplotypeusingthebinnedreads,excludingunclassifiedreads.ThecontigswerepolishedwithtworoundsofArrowpolishingusingthebinnedreads,andscaffoldedfollowingthev1.0pipelinewithnopurging.AdditionalscaffoldingroundswithBionano(s4)andHi-Cwereapplied.Scaffoldswererenamedaccordingtotheprimaryscaffoldassemblyofthesameindividual(s5),withsexchromosomesgroupedasZinthepaternalassemblyandWinthematernalassemblyfollowingsyntenytotheZchromosomefromthecuratedmalezebrafinchVGPassembly.TworoundsofSRpolishingwereappliedusinglinkedreads,bymappingonbothhaplotypes.Afterhaplotypeswitcheswerediscovered,additionalroundsofpolishingwereappliedusingbinnedlinkedreads(SupplementaryMethods).MitochondrialgenomeassemblySimilartootherrecentmethods93,94,wedevelopedareference-guidedMTassemblypipeline.MTreadsintherawCLRdatawereidentifiedbymappingthewholereadsettoanexistingreferencesequenceofthespecificspeciesorofcloselyrelatedspeciesusingBlasr.FilteredmtDNACLRswereassembledintoasinglecontigusingCanuv1.8,polishedwithArrowusingCLRandthenFreeBayesv1.0.2togetherwithbcftoolsv1.9usingshortreadsfromthe10XGdata(ExtendedDataFig.3c).Theoverlappingsequencesattheendsofthecontigweretrimmed,andtheremainingcontigsequencecircularized.ThemitoVGPpipelineismadeavailableathttps://github.com/VGP/vgp-assembly/tree/master/mitoVGP.AmoredetailedprotocoldescriptionoftheassemblypipelineandnewdiscoveriesfromtheMTassembliesarepublishedelsewhere33.CurationTheVGPgenomeassemblypipelineproduceshighqualityassemblies,yetnoautomatedmethodtodateisfreefromtheproductionoferrors,especiallyduringthescaffoldingstages.Tominimizetheimpactoftheremainingalgorithmicshortcomings,wesubjectedallassembliestorigorousmanualcuration.Alldatageneratedforaspeciesinthisstudyandotherpubliclyavailabledata(forexample,geneticmaps,genesetsandgenomeassembliesofthesameorcloselyrelatedspecies)werecollated,alignedtotheprimaryassemblyandanalysedingEVAL95(https://vgp-geval.sanger.ac.uk/index.html),visualizingdiscordancesinafeaturebrowserandissuelists.Inparallel,Hi-CdataweremappedtotheprimaryassemblyandvisualizedusingJuicebox96and/orHiGlass97.Withthesedata,genomecuratorsidentifiedmis-joins,missedjoinsandotheranomalies,andcorrectedtheprimaryassemblyaccordingly.Nochangewasmadewithoutunambiguousevidencefromavailabledatatypes;forexample,aHi-CsuggestedjoinwouldnotbemadeunlesssupportedbyBioNanomaps,long-readdata,orgenealignments.Whensequencingtheheterogameticsex,weidentifiedsexchromosomesbasedonhalfcoverage,homologyalignmentstosexchromosomesinotherspecies,andthepresenceofsexchromosome-specificgenes.ContaminationremovalAsuccessionofsearcheswasusedtoidentifypotentialcontaminantsinthegeneratedassemblies.1)AmegaBLAST98searchagainstadatabaseofcommoncontaminants(ftp://ftp.ncbi.nlm.nih.gov/pub/kitts/contam_in_euks.fa.gz)requiringe ≤ 1 × 10−4,reportingmatches with≥98%sequenceidentityandmatchlength50–99bp, ≥94%andmatchlength100–199bp,or ≥90%andmatchlength200bporabove.2)Avecscreen(https://www.ncbi.nlm.nih.gov/tools/vecscreen/)searchagainstadatabaseofadaptorsequences(ftp://ftp.ncbi.nlm.nih.gov/pub/kitts/adaptors_for_screening_euks.fa)3)Aftersoft-maskingrepeatsusingWindowmasker75,amegaBLASTsearchagainstchromosome-levelassembliesfromRefSeqrequiringe ≤ 1 × 10−4,matchscore≥100,andsequenceidentity ≥98%;regionsmatchinghighlyconservedrDNAswereignored.Manualinspectionoftheresultswasnecessarytodifferentiatecontaminationfromconservationand/orhorizontalgenetransfer.Adaptorsequencesweremasked;othercontaminantsequenceswereremoved.AssemblieswerealsocheckedforrunsofNsattheendsofscaffolds,createdasartefactsoftheiterativescaffoldingprocess,andwhenfoundtheyweretrimmed.OrganellegenomesTheseweredetectedbyamegaBLASTsearchagainstadatabaseofknownorganellegenomesrequiringe ≤ 1 × 10−4,sequenceidentity ≥90%,andmatchlength ≥500;thedatabasesareavailableatftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/mito.nt.gzandftp://ftp.ncbi.nlm.nih.gov/refseq/release/plastid/*genomic.fna.gz.Onlyscaffoldsconsistingentirelyoforganellesequenceswereassumedtobeorganellegenomes,andreplacedbythegenomefromtheseparateorganelleassemblypipeline.OrganellematchesembeddedinnuclearsequencesthatwerefoundtobeNuMTswerekept.FalseduplicationremovalRetainedfalseduplicationswereidentifiedusingPurge_Haplotigs13runeitherafterscaffoldingandpolishing(Anna’shummingbird,kākāpō,malezebrafinch,femalezebrafinch,platypus,palespear-nosedbat,andgreaterhorseshoebat)oronthec1beforescaffolding(two-linedcaecilian,fliercichlid,Canadalynx,andGoode’sthornscrubtortoise).SubsequentmanualcurationidentifiedadditionalhaplotypicduplicationsforthelistedassembliesandalsothosethatwerenottreatedwithPurge_Haplotigs(Easternhappy,climbingperch,zig-zageel).Theevidenceusedincludedreadcoverage,sequenceself-comparison,transcriptalignments,BionanomapalignmentsandHi-C2Dmaps,allconfirmingthesuperfluousnatureofoneallele.Theidentifiedhaplotypeduplicationsweremovedfromtheprimarytothealternateassembly.ChromosomeassignmentForascaffoldtobeannotatedasachromosome,weusedevidencefromHi-CaswellasgeneticlinkageorFISHkaryotypemappingwhenavailable.ForHi-Cevidence,weconsideredascaffoldasacompletechromosome(albeitwithgaps)whentherewasaclearunbrokendiagonalintheJuiceboxorHiGlassplotsforthatscaffoldandnootherlargescaffoldsthatcouldbejoinedtothatsamescaffold;ifpresentandnounambiguousjoinwaspossible,wenameditasanunlocalizedscaffoldforthatchromosome.Whenwecouldnotfindevidenceofacompletechromosome,wekeptthescaffoldnumberforitsname.Wenamedallevidence-validatedscaffoldsaschromosomesdowntothesmallestHi-Cboxunitresolutionallowedwiththesecharacteristics.Whentherewasanestablishedchromosometerminologyforagivenspeciesorsetofspecies,weusetheestablishedterminologyexceptwhenournewassembliesrevealederrorsintheolderassembly,suchasscaffold/chromosomefusions,fissions,rearrangements,andnon-chromosomenames.Forspecieswithoutanestablishedchromosometerminology,wenamedthescaffoldsaschromosomesnumbers1,2,3…,indescendingorderofscaffoldsize.Forthesexchromosomes,weusedthelettersXandYformammalsandZandWforbirds.UsingcomparativegenomicstoassessassemblystructureIncaseswhereahigh-qualitychromosome-levelgenomewasavailableforacloselyrelatedspecies,comparativegenomeanalysiswasperformed.Thepolishedprimaryassembly(t3.p)wasmappedtotherelatedgenomeusingMashMap286with--pi75-s300000.Thenumberofchromosomaldifferenceswasidentifiedusingacustomscriptavailableathttps://github.com/jdamas13/assembly_comparison.Thisresultedintheidentificationof~60to~450regionsforeachgenomeassemblyflankingputativemisassembliesorlineage-specificgenomerearrangements.Toidentifywhichwererealmisassemblies,theidentifieddiscrepancieswerecommunicatedtothecurationteamformanualverification(seeabove).Toidentifyanypossibleremainingmis-joins,eachcuratedavianandmammalianassemblywascomparedwiththezebrafinch(taeGut2)orhuman(hg38)genomes,respectively.PairwisealignmentsbetweeneachoftheVGPassembliesandthecladereferenceweregeneratedwithLastZ99(version1.04)usingthefollowingparameters:C = 0E = 30H = 2000K = 3000L = 2200O = 400.ThepairwisealignmentswereconvertedintotheUCSC‘chain’and‘net’formatswithaxtChain(parameters:-minScore = 1000-verbose = 0-linearGap = medium)followedbychainAntiRepeat,chainSort,chainPreNet,chainNetandnetSyntenic,allwithdefaultparameters100.Pairwisesyntenyblocksweredefinedusingmaf2synteny101at100-,300-,and500-kbresolutions.Evolutionarybreakpointregionsweredetectedandclassifiedusinganadhocstatisticalapproach102.Thisanalysisidentified2to90genomicregionsperassemblythatcouldbeflankingmisassemblies,lineage-specificchromosomerearrangements,orreference-specificchromosomerearrangements(116inthehumanand26inthezebrafinch).Determiningtheunderlyingcauseforeachoftheflaggedregionswillneedfurtherverification.AllalignmentsareavailableforvisualizationattheEvolutionHighwaycomparativechromosomebrowser(http://eh-demo.ncsa.illinois.edu/vgp/).AnnotationNCBIandEnsemblannotationpipelineusedinthisstudyaredescribedinthe SupplementaryMethods.EvaluationDetailedmethodsforothertypesofevaluation,includingBUSCOruns,mis-joinandmissed-joinidentification,reliableblocks,collapsedrepeats,telomeres,RNA-seqandATAC–seqmapping,andfalsegeneduplicationsareinthe SupplementaryMethods.Nostatisticalmethodswereusedtopredeterminesamplesize,theexperimentswerenotrandomized,andtheinvestigatorswerenotblindedtogroupduringexperimentsandoutcomeassessment.ReportingsummaryFurtherinformationonresearchdesignisavailableinthe NatureResearchReportingSummarylinkedtothispaper. Dataavailability Allrawdata,intermediateandfinalassembliesarepubliclyavailableviaGenomeArk(https://vgp.github.io/genomeark),archivedonNCBI/EBIBioProjectunderaccessionPRJNA489243withannotations,andbrowsableontheUCSCGenomeBrowser(https://hgdownload.soe.ucsc.edu/hubs/VGP/).ThefinalprimaryassemblyfromtheautomatedpipelinebeforecurationisbrowsableongEVAL(https://vgp-geval.sanger.ac.uk)withallfourrawdatamappings.TheVGPassemblypipelineisavailableasastand-alonepipeline(https://github.com/VGP/vgp-assembly)aswellasaworkflowonDNAnexus(https://platform.dnanexus.com/).AVGP-specificassemblyhubportalintheU.C.SantaCruzbrowserisavailableasagatewaytoaccessallVGPgenomeassembliesandannotations(https://hgdownload.soe.ucsc.edu/hubs/VGP). Codeavailability AllcodesusedintheVGPAssemblyPipelineandtheVGPTrioPipelinearepubliclyavailableathttps://github.com/VGP/vgp-assembly/tree/master/pipeline. 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dResearch(01IS18026C).ComplementarysequencingsupportfortheAnna’shummingbirdandseveralgenomeswasprovidedbyPacificBiosciences,BionanoGenomics,DovetailGenomics,ArimaGenomics,PhaseGenomics,10XGenomics,NRGene,OxfordNanoporeTechnologies,Illumina,andDNAnexus.AllothersequencingandassemblywereconductedattheRockefellerUniversity,SangerInstitute,andMaxPlanckInstituteDresdengenomelabs.PartofthisworkusedthecomputationalresourcesoftheNIHHPCBiowulfcluster(https://hpc.nih.gov).WeacknowledgefundingfromtheWellcomeTrust(108749/Z/15/Z)andtheEuropeanMolecularBiologyLaboratory.WethankLeComitéScientifiqueRégionalduPatrimoineNaturelandDirectiondel’Environnement,del’AménagementetduLogement,Guyanneforresearchapprovalsandexportpermits.AuthorinformationAuthornotesTheseauthorscontributedequally:ArangRhie,ShaneA.McCarthy,OlivierFedrigoTheseauthorsjointlysupervisedthiswork:KerstinHowe,EugeneW.Myers,RichardDurbin,AdamM.Phillippy,ErichD.JarvisAffiliationsGenomeInformaticsSection,ComputationalandStatisticalGenomicsBranch,NationalHumanGenomeResearchInstitute,NationalInstitutesofHealth,Bethesda,MD,USAArangRhie, SergeyKoren, BrianP.Walenz & AdamM.PhillippyDepartmentofGenetics,UniversityofCambridge,Cambridge,UKShaneA.McCarthy, IlianaBista, DengfengGuan & RichardDurbinWellcomeSangerInstitute,Cambridge,UKShaneA.McCarthy, WilliamChow, IlianaBista, MichelleSmith, MilanMalinsky, ZeminNing, YingSims, JoannaCollins, SarahPelan, JamesTorrance, AlanTracey, JonathanWood, KerstinHowe & RichardDurbinVertebrateGenomeLab,TheRockefellerUniversity,NewYork,NY,USAOlivierFedrigo, GiulioFormenti, BettinaHaase, JacquelynMountcastle, SadyePaez & ErichD.JarvisTheGenomeCenter,UniversityofCaliforniaDavis,Davis,CA,USAJoanaDamas & HarrisA.LewinLaboratoryofNeurogeneticsofLanguage,TheRockefellerUniversity,NewYork,NY,USAGiulioFormenti, GregoryL.Gedman, LindseyJ.Cantin, SadyePaez, MatthewT.Biegler, ConstantinaTheofanopoulou & ErichD.JarvisLeibnizInstituteforZooandWildlifeResearch,DepartmentofEvolutionaryGenetics,Berlin,GermanyMarcelaUliano-SilvaBerlinCenterforGenomicsinBiodiversityResearch,Berlin,GermanyMarcelaUliano-SilvaDNAnexusInc.,MountainView,CA,USAArkarachaiFungtammasan, MariaSimbirsky & BrettT.HanniganInterdisciplinaryPrograminBioinformatics,SeoulNationalUniversity,Seoul,RepublicofKoreaJuwanKim, ChulLee & HeebalKimDepartmentofAgriculturalBiotechnologyandResearchInstituteofAgricultureandLifeSciences,SeoulNationalUniversity,Seoul,RepublicofKoreaByungJuneKo & HeebalKimUniversityofSouthernCalifornia,LosAngeles,CA,USAMarkChaisson & RobelE.DagnewNationalCenterforBiotechnologyInformation,NationalLibraryofMedicine,NIH,Bethesda,MD,USAFrancoiseThibaud-Nissen, JinnaHoffman, PatrickMasterson & KarenClarkEuropeanMolecularBiologyLaboratory,EuropeanBioinformaticsInstitute,WellcomeGenomeCampus,Hinxton,UKLeanneHaggerty, FergalMartin, KevinHowe & PaulFlicekMaxPlanckInstituteofMolecularCellBiologyandGenetics,Dresden,GermanySylkeWinkler, MartinPippel, EkaterinaOsipova & EugeneW.MyersDRESDEN-conceptGenomeCenter,Dresden,GermanySylkeWinklerNovogene,Durham,NC,USAJasonHowardNeurogeneticsofVocalCommunicationGroup,MaxPlanckInstituteforPsycholinguistics,Nijmegen,TheNetherlandsSonjaC.VernesDondersInstituteforBrain,CognitionandBehaviour,Nijmegen,TheNetherlandsSonjaC.VernesSchoolofBiology,UniversityofStAndrews,StAndrews,UKSonjaC.VernesUniversityofMassachusettsCooperativeFishandWildlifeResearchUnit,Amherst,MA,USATanyaM.LamaSchoolofBiologicalScience,TheEnvironmentInstitute,UniversityofAdelaide,Adelaide,SouthAustralia,AustraliaFrankGrutznerBondLifeSciencesCenter,UniversityofMissouri,Columbia,MO,USAWesleyC.WarrenDepartmentofBiology,EastCarolinaUniversity,Greenville,NC,USAChristopherN.BalakrishnanUQGenomics,UniversityofQueensland,Brisbane,Queensland,AustraliaDaveBurtDepartmentofBiologicalSciences,ClemsonUniversity,Clemson,SC,USAJuliaM.GeorgeTheGeneticRescueFoundation,Wellington,NewZealandDavidIornsKākāpōRecovery,DepartmentofConservation,Invercargill,NewZealandAndrewDigby & DarylEasonDepartmentofZoology,UniversityofOtago,Dunedin,NewZealandBruceRobertsonUniversityofArizonaGeneticsCore,Tucson,AZ,USATaylorEdwardsDepartmentofLifeSciences,NaturalHistoryMuseum,London,UKMarkWilkinsonSchoolofNaturalSciences,BangorUniversity,Gwynedd,UKGeorgeTurnerDepartmentofBiology,UniversityofKonstanz,Konstanz,GermanyAxelMeyer, AndreasF.Kautt, PaoloFranchini & RobertH.S.KrausDepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,Cambridge,MA,USAAndreasF.KauttDepartmentofMarineandEnvironmentalSciences,NortheasternUniversityMarineScienceCenter,Nahant,MA,USAH.WilliamDetrichIIIDepartmentofBiology,UniversityofAntwerp,Antwerp,BelgiumHannesSvardalNaturalisBiodiversityCenter,Leiden,TheNetherlandsHannesSvardalInstituteofBiology,Karl-FranzensUniversityofGraz,Graz,AustriaMaximilianWagnerFloridaMuseumofNaturalHistory,UniversityofFlorida,Gainesville,FL,USAGavinJ.P.NaylorCenterforSystemsBiology,Dresden,GermanyMartinPippel, EkaterinaOsipova & EugeneW.MyersZoologicalInstitute,UniversityofBasel,Basel,SwitzerlandMilanMalinskyTag.bio,SanFrancisco,CA,USAMarkMooneyUCSantaCruzGenomicsInstitute,UniversityofCalifornia,SantaCruz,CA,USATrevorPesout, RichardE.Green, ErikGarrison, HiramClawson, MarkDiekhans, LuisNassar, BenedictPaten & DavidHausslerSanDiegoZooGlobal,Escondido,CA,USAMarlysHouck, AnnMisuraca & OliverA.RyderPacificBiosciences,MenloPark,CA,USASarahB.Kingan, RichardHall, ZevKronenberg, IvanSović, ChristopherDunn & JonasKorlachDigitalBioLogic,Ivanić-Grad,CroatiaIvanSovićBionanoGenomics,SanDiego,CA,USAAlexHastie & JoyceLeeArimaGenomics,SanDiego,CA,USASiddarthSelvarajDovetailGenomics,SantaCruz,CA,USARichardE.Green & JayGhuryeIndependentResearcher,SantaCruz,CA,USANicholasH.PutnamCNAG-CRG,CentreforGenomicRegulation,BarcelonaInstituteofScienceandTechnology,Barcelona,SpainIvoGutUniversitatPompeuFabra,Barcelona,SpainIvoGutDepartmentofComputerScience,UniversityofMarylandCollegePark,CollegePark,MD,USAJayGhuryeSchoolofComputerScienceandTechnology,CenterforBioinformatics,HarbinInstituteofTechnology,Harbin,ChinaDengfengGuanDepartmentofPsychology,InstituteforMindandBiology,UniversityofChicago,Chicago,IL,USASarahE.LondonDepartmentofGeneticsandBiochemistry,ClemsonUniversity,Clemson,SC,USADavidF.ClaytonDepartmentofBehavioralNeuroscience,OregonHealthandScienceUniversity,Portland,OR,USAClaudioV.Mello, SamanthaR.Friedrich & PeterV.LovellMaxPlanckInstituteforthePhysicsofComplexSystems,Dresden,GermanyEkaterinaOsipovaMonashUniversityMalaysiaGenomicsFacility,SchoolofScience,SelangorDarulEhsan,MalaysiaFarooqO.Al-AjliTropicalMedicineandBiologyMultidisciplinaryPlatform,MonashUniversityMalaysia,SelangorDarulEhsan,MalaysiaFarooqO.Al-AjliQatarFalconGenomeProject,Doha,QatarFarooqO.Al-AjliDepartmentofBiosciences,UniversityofMilan,Milan,ItalySimonaSecomandieGnome,Inc.,Seoul,RepublicofKoreaHeebalKim & WooriKwakLOEWECentreforTranslationalBiodiversityGenomics,Frankfurt,GermanyMichaelHillerSenckenbergResearchInstitute,Frankfurt,GermanyMichaelHillerGoethe-University,FacultyofBiosciences,Frankfurt,GermanyMichaelHillerBGI-Shenzhen,Shenzhen,ChinaYangZhouDepartmentofBiology,PennsylvaniaStateUniversity,UniversityPark,PA,USARobertS.Harris & KaterynaD.MakovaCenterforMedicalGenomics,PennsylvaniaStateUniversity,UniversityPark,PA,USAKaterynaD.Makova & PaulMedvedevCenterforComputationalBiologyandBioinformatics,PennsylvaniaStateUniversity,UniversityPark,PA,USAKaterynaD.Makova & PaulMedvedevDepartmentofComputerScienceandEngineering,PennsylvaniaStateUniversity,UniversityPark,PA,USAPaulMedvedevDepartmentofBiochemistryandMolecularBiology,PennsylvaniaStateUniversity,UniversityPark,PA,USAPaulMedvedevHoonygen,Seoul,KoreaWooriKwakDepartmentofMigration,MaxPlanckInstituteofAnimalBehavior,Radolfzell,GermanyRobertH.S.KrausDepartmentofBiologicalSciences,UniversidaddelosAndes,Bogotá,ColombiaAndrewJ.CrawfordCenterforEvolutionaryHologenomics,TheGLOBEInstitute,UniversityofCopenhagen,Copenhagen,DenmarkM.ThomasP.GilbertUniversityMuseum,NTNU,Trondheim,NorwayM.ThomasP.GilbertChinaNationalGenebank,BGI-Shenzhen,Shenzhen,ChinaGuojieZhangVillumCenterforBiodiversityGenomics,SectionforEcologyandEvolution,DepartmentofBiology,UniversityofCopenhagen,Copenhagen,DenmarkGuojieZhangStateKeyLaboratoryofGeneticResourcesandEvolution,KunmingInstituteofZoology,ChineseAcademyofSciences,Kunming,ChinaGuojieZhangCenterforExcellenceinAnimalEvolutionandGenetics,ChineseAcademyofSciences,Kunming,ChinaGuojieZhangInstituteofMolecularandCellBiology,A*STAR,Biopolis,Singapore,SingaporeByrappaVenkateshCentreforBiodiversity,RoyalOntarioMuseum,Toronto,Ontario,CanadaRobertW.MurphySmithsonianConservationBiologyInstitute,CenterforSpeciesSurvival,NationalZoologicalPark,Washington,DC,USAKlaus-PeterKoepfli & WarrenE.JohnsonDepartmentofEcologyandEvolutionaryBiology,UniversityofCaliforniaSantaCruz,SantaCruz,CA,USABethShapiro & DavidHausslerHowardHughesMedicalInstitute,ChevyChase,MD,USABethShapiro & ErichD.JarvisTheWalterReedBiosystematicsUnit,MuseumSupportCenterMRC-534,SmithsonianInstitution,Suitland,MD,USAWarrenE.JohnsonWalterReedArmyInstituteofResearch,SilverSpring,MD,USAWarrenE.JohnsonDepartmentofBiologicalSciences,EarlhamInstitute,UniversityofEastAnglia,Norwich,UKFedericaDiPalmaInstituteofEvolutionaryBiology(UPF-CSIC),PRBB,Barcelona,SpainTomasMarques-BonetCatalanInstitutionofResearchandAdvancedStudies(ICREA),Barcelona,SpainTomasMarques-BonetCentreforGenomicRegulation(CRG),BarcelonaInstituteofScienceandTechnology(BIST),Barcelona,SpainTomasMarques-BonetInstitutCatalàdePaleontologiaMiquelCrusafont,UniversitatAutònomadeBarcelona,Barcelona,SpainTomasMarques-BonetSchoolofBiologyandEnvironmentalScience,UniversityCollegeDublin,Dublin,IrelandEmmaC.TeelingDepartmentofComputerScience,TheUniversityofIllinoisatUrbana-Champaign,Urbana,IL,USATandyWarnowSchoolofLifeScience,LaTrobeUniversity,Melbourne,Victoria,AustraliaJenniferMarshallGravesDepartmentofEvolution,Behavior,andEcology,UniversityofCaliforniaSanDiego,LaJolla,CA,USAOliverA.RyderLaboratoryofGenomicsDiversity-CenterforComputerTechnologies,ITMOUniversity,St.Petersburg,RussianFederationStephenJ.O’BrienGuyHarveyOceanographicCenter,HalmosCollegeofNaturalSciencesandOceanography,NovaSoutheasternUniversity,FortLauderdale,FL,USAStephenJ.O’BrienDepartmentofEvolutionandEcology,UniversityofCaliforniaDavis,Davis,CA,USAHarrisA.LewinJohnMuirInstitutefortheEnvironment,UniversityofCaliforniaDavis,Davis,CA,USAHarrisA.LewinFacultyofComputerScience,TechnicalUniversityDresden,Dresden,GermanyEugeneW.MyersAuthorsArangRhieViewauthorpublicationsYoucanalsosearchforthisauthorin 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PubMed GoogleScholarContributionsWrotethepaperandco-coordinatedthestudy:A.R.,E.D.J.,A.M.P.,R.D.,E.W.M.,KerstinHowe,S.A.M.,O.F.Coordinationwithvendors:J.Korlach,S.Selvaraj,R.E.G.,A.H.,M.Mooney.Collectedsamples:M.T.P.G.,W.E.J.,R.W.M.,G.Z.,B.V.,M.T.B.,J.Howard,S.C.V.,T.M.L.,F.G.,W.C.W.,D.B.,J.M.George,M.T.B.,D.I.,A.D.,D.E.,B.R.,T.E.,M.Wilkinson,G.T.,A.Meyer,A.F.K.,P.Franchini,H.W.D.,H.S.,M.Wagner,G.J.P.N.,R.D.,E.D.J.,E.C.T.,R.H.S.K.Generatedgenomedata:O.F.,I.B.,M.Smith,B.H.,J.M.,S.W.,C.B.,A.Meyer,A.F.K.,P.Franchini,I.G.,D.F.C.,C.V.M.Generatedgenomeassemblies:A.R.,S.A.M.,S.K.,M.P.,S.B.K.,R.H.,J.G.,Z.N.,J.L.,B.P.W.,M.Malinsky.Generated/modifiedsoftware:S.K.,A.R.,S.B.K.,R.H.,Z.K.,J.Korlach,I.S.,C.D.,Z.N.,A.H.,J.L.,J.G.,E.G.,C.V.M.,S.R.F.,N.H.P.Pipelinedevelopment:A.R.,S.A.M.,G.F.,S.K.,M.U.-S.,A.F.,M.Simbirsky,B.T.H.,T.P.,M.P.,E.W.M.,R.D.,A.M.P.GeneratedMTassemblies:G.F.,J.Korlach.Curation:KerstinHowe,W.C.,Y.S.,J.C.,S.Pelan,J.T.,A.T.,J.W.,Y.Z.,J.D.,H.A.L.Sexchromosomes:Y.Z.,R.S.H.,K.D.M.,P.Medvedev,J.M.Graves.Hummingbirdkaryotypeanalyses:M.Houck,A.Misuraca,M.P.,E.W.M.,E.D.J.Annotation:F.T.-N.,L.H.,J.Hoffman,P.Masterson,K.C.,F.M.,KevinHowe,P.Flicek,D.B.Evaluationanalysis:A.R.,J.D.,M.U.-S.,J.Kim,C.L.,B.J.K.,M.C.,G.L.G.,L.J.C.,F.T.-N.,L.H.,J.M.George,J.G.,R.E.D.,D.G.,S.E.L.,D.F.C.,C.V.M.,S.R.F.,P.V.L.,E.O.,F.O.A.-A.,S.Secomandi,C.T.,M.Hiller,H.K.,KerstinHowe,E.W.M.,R.D.,A.M.P.,E.D.J.Biologicalfindings:J.D.,J.Kim,C.L.,B.J.K.,G.L.G.,L.J.C.,H.A.L.,A.R.,E.D.J.Dataavailability:A.R.,S.A.M.,W.C.,A.F.,S.Paez,M.Simbirsky,B.T.H.,B.P.W.,W.K.,H.C.,M.D.,L.N.,B.P.,A.M.P.,E.D.J.G10Kcouncil,founders,andcoordinationofVGP:T.M.-B.,A.J.C.,F.D.P.,R.D.,M.T.P.G.,E.D.J.,K.-P.K.,H.A.L.,R.W.M.,E.W.M.,E.C.T.,B.V.,G.Z.,A.M.P.,S.Paez,J.M.Graves,O.A.R.,D.H.,S.J.O.,T.W.andB.S.Allauthorsreviewedthemanuscript.CorrespondingauthorsCorrespondenceto KerstinHowe,EugeneW.Myers,RichardDurbin,AdamM.PhillippyorErichD.Jarvis.Ethicsdeclarations Competinginterests Duringthecontributingperiod,B.T.H.,M.Simbirsky,A.F.andM.MooneywereemployeesofDNAnexusInc.S.B.K.,R.H.,Z.K.,J.Korlach,I.S.andC.D.werefull-timeemployeesatPacificBiosciences,acompanydevelopingsingle-moleculelongreadsequencingtechnologies.R.E.G.,N.H.P.,andJ.G.wereaffiliatedwithDovetailGenomics,acompanydevelopinggenomeassemblytools,includingHi-C.I.G.wasaffiliatedwithOxfordNanoporeTechnologies,acompanygeneratinglongreadsequencingtechnologies.A.H.andJ.LwereemployeesofBionanoGenomics,acompanydevelopingopticalmapsforgenomeassembly.S.SelvarajwasanemployeeofArimaGenomics,acompanydevelopingHi-Cdataforgenomeassemblies.R.D.isascientificadvisoryboardmemberofDovetailInc.P.FlicekisamemberoftheScientificAdvisoryBoardsofFabricGenomics,Inc.,andEagleGenomics,Ltd.H.C.receivesroyaltiesfromthesaleofUCSCGenomeBrowsersourcecode,LiftOver,GBiB,andGBiClicensestocommercialentities.S.K.hasreceivedtravelfundstospeakatsymposiaorganizedbyOxfordNanopore.M.D.andL.N.receiveroyaltiesfromlicensingofUCSCGenomeBrowser.ForW.E.J.,thecontenthereisnottobeconstruedastheviewsoftheDAorDOD.Allotherauthorsdeclarenocompetinginterests. AdditionalinformationPeerreviewinformationNaturethanksMichaelSchatz,JustinZookandtheother,anonymous,reviewer(s)fortheircontributiontothepeerreviewofthiswork.Peerreviewerreportsareavailable.Publisher’snoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.ExtendeddatafiguresandtablesExtendedDataFig.1AssessmentofcompletenessoftheAnna’shummingbirdassembly.a,b,StepsandNG50continuityvaluesoftheVGPassemblypipelinethatgavethehighestqualityassemblyforAnna’shummingbird(a)andCanadalynx(b)inthisstudy.ThespecificstepsareoutlinedfurtherinExtendedDataFig.2a,andMethods.c,Whole-genomealignmentofCLR(red),linkedreads(green),opticalmaps(blue),andHi-Creads(purple)oftheAnna’shummingbird,alongwithtelomeremotif(TTAGGGanditsreversecomplement,yellow)andgaps(grey)usingAssetsoftware103.Foreachdatatype,thefirstrowshowsthemappedcoverage,andthesecondshowsthenumberofcountsoflowcoverageorsignsofcollapsedrepeats.Largerchromosomalscaffolds(1–19)havefewergapsandlowcoverageorcollapsedregionscomparedwiththemicrochromosomes(20–33).Chromosomes14,15and19oftheAnna’shummingbirdwerethemoststructurallyreliablescaffolds,havingonlyonegapeachwithnolow-supportregions.Wedefinedreliableblocksasthosesupportedbyatleasttwotechnologies.Reliableblocksexcludedregionswithstructuralassemblyerrors,suchascollapsedrepeatsorunresolvedsegmentalduplications.Low-supportregionsarethosewherethereliableblocksrowhasapeak.ExtendedDataFig.2VGPassemblypipelineappliedacrossmultiplespecies.a,Iterativeassemblypipelineofsequencedatatypes(colouredasinb)withincreasingchromosomaldistance.Thinbars,sequencereads;thickblackbars,assembledcontigs;blackbarswithspaceandarcinglinks,scaffolds;greybars,gapsplacedbyprevioussteps;thickredborder,trackingofanexamplecontiginthepipeline.Thecurationstepshowsanexampleofamis-assemblybreakidentifiedbysequencecoverage(grey,left)andanexampleofaninversionerror(right)detectedbytheopticalmap.b,Intra-moleculelengthdistributionofthefourdatatypesusedtogeneratetheassembliesof16vertebratespecies,weightedbythefractionofbasesineachlengthbin(logscaled).Moleculelengthabove1 kbwasmeasuredfromreadlengthforCLR,estimatedmoleculecoverageforlinkedreads,rawmoleculelengthforopticalmaps,andinteractiondistanceforHi-Creads.Foreachspecies,thefragmentlengthdistributionofeachdatatypewassimilartothosefortheAnna’shummingbird,withdifferencesprimarilyinfluencedbytissuetype,preservationmethod,andcollectionorstorageconditions(unpublisheddata).ExtendedDataFig.3Flowchartsofassemblypipelinesusedtogeneratehigh-qualityassembliesinthisstudy.a,StandardVGPassemblypipelinewhensequencingdataofoneindividual,thatgeneratedthehighestqualityassemblies:generateprimarypseudo-haplotypeandalternatehaplotypecontigswithCLRusingFALCON-Unzip17;generatescaffoldswithlinkedreadsusingScaff10x74;breakmis-joinsandfurtherscaffoldwithopticalmapsusingSolve87;generatechromosome-scalescaffoldswithHi-CreadsusingSalsa279;fillingapsandpolishbase-errorswithCLRusingArrow(PacificBioSciences);performtwoormoreroundsofshort-readpolishingwithlinkedreadsusingFreeBayes85;andperformexpertmanualcurationtocorrectpotentialassemblyerrorsusinggEVAL25,95b,StandardVGPtrioassemblypipelinewhenDNAisavailableforachildandparents20.Dashedlineindicatesthattheotherhaplotypewentthroughthesamestepsbeforecuration.Inadditiontothecuratedassembliesofbothhaplotypes,arepresentativehaplotypewithbothsexchromosomesissubmitted.c,Mitochondrialassemblypipeline.Figurekeyappliestoa–c.Stepsnewlyintroducedinv1.5–v1.6arehighlightedinlightblue.c,contigs;p,purgedfalseduplicationsfromprimarycontigs;q,purgedalternatecontigs;s,scaffolds;t,polishedscaffolds.Furtherdetailsandinstructionsareavailableelsewhere33andathttps://github.com/VGP/vgp-assembly.ExtendedDataFig.4Relationshipbetweencollapsesandgenomiccharacteristics.a,Correlationbetweenthetotalnumberofcollapsesandpercentagerepeatcontentestimatedinthesubmittedcuratedversionsofn=17genomesfrom16species.b,CorrelationbetweentotalnumberofbasesincollapsedregionsperGbandrepeatcontent.c,CorrelationbetweentotalmissingbasescollapsedperGbandrepeatcontent.d,Correlationbetweentotalnumberofgenes(codingandnon-coding)inthecollapsedregionsandrepeatcontent.e,Lackofcorrelationbetweentheaveragecollapsedsizeandrepeatcontent.f,Lackofcorrelationbetweenthetotalnumberofcollapsesandpercentageheterozygosity.g,LackofcorrelationbetweenthetotalnumberofcollapsesperGbandgenomesize.Genomesize,heterozygosity,andrepeatcontentwereestimatedfrom31-mercountsusingGenomeScope71.Reportedareadjustedr2andPvaluesfromF-statistics.h,CumulativecollapsedbasesperGbineachcollapseandpercentagerepeatmasked.Eachcircleiscolouredbyspecieswithitssizerelativetothelengthofthe collapseasitappearsintheassembly.Collapsesabovethehorizontalbar(>90%)arefurtherclassifiedascollapsedhigh-copyrepeats,andthosebelowthehorizontalbarareclassifiedassegmentalduplications(low-copyrepeats).i,Majorrepeattypesincollapsedhigh-copyrepeats.MostoftherepeatsweremaskedonlywithWindowMasker75,withnoannotationavailablebyRepeatMasker104.j,Minorrepeattypesincollapsedrepeats.Thisisabreakdownoftherepeatscategorizedas‘Others’ini,owingtothesmallerscale.Barcoloursiniandjareasinh.Notesmallerscaleinjcomparedwithi.Collapsedsatellitearrayswerealmostexclusivelyfoundintheplatypus,comprising~2.5Mb.Collapsedsimplerepeatswerethemajorsourceinthethornyskate(~400kb).TherewasahigherproportionofLTRsinbirds,LINEsandSINEsinmammals,andDNArepeatsintheamphibian.Amongthegenesinthecollapses,manywererepetitiveshortnon-codingRNAs.PvaluesfromF-statistics.ExtendedDataFig.5Falseduplicationmechanismsingenomeassembly.a,Falseheterotype(haplotype)duplicationsoccurswhenmoredivergentsequencereadsfromeachhaplotypeA(blue)andB(red)(maternalandpaternal)formgreaterdivergentpathsintheassemblygraph(bubbles),whilenearlyidenticalhomozygoussequences(black)becomecollapsed.Whentheassemblygraphisproperlyformedandcorrectlyresolved(greenarrow),oneofthehaplotype-specificpaths(redorblue)ischosenforbuildinga‘primary’pseudo-haplotypeassemblyandtheotherissetapartasan‘alternate’assembly.Whenthegraphisnotcorrectlyresolved(purplearrow),oneoffourtypesofpatternareformedinthecontigsandsubsequentscaffolds.Dependingonthesupportingevidence,thescaffoldereitherkeepsthesehaplotypecontigsonseparatescaffoldsorbringsthemtogetheronthesamescaffold,oftenseparatedbygaps:1.Separatecontigs:bothcontigsareretainedintheprimarycontigset,anerroroftenobservedwhenhaplotype-specificsequencesarehighlydiverged.2.Flankingcontigs:theassemblygraphispartiallyformed,connectingthehomozygoussequenceofthe5′sidetoonehaplotype(blue)andthe3′sidetotheotherhaplotype(red).3.Partialflankingcontigs:onlyonehaplotype(blue)flanksonesideofthehomozygoussequence.4.Failedconnectingofcontigs:allhaplotypesequencesfailtoproperlyconnecttoflankinghomozygoussequences.b,Falsehomotypeduplicationsoccurwhereasequencefromthesamegenomiclocusisduplicated,andareoftwotypes:1.Overlappingsequencesatcontigboundaries:incurrentoverlap-layout-consensusassemblers,branchingsequencesinassemblygraphsthatarenotselectedastheprimarypathhaveasmalloverlappingsequence(purple),dovetailingtotheprimarypathwhereitoriginatedabranch.Thesizeoftheduplicatedsequenceisoftenthelengthofacorrectedread.Subsequentscaffoldingresultsintandemduplicatedsequenceswithagapbetween.2.Under-collapsedsequences:sequencingerrorsinreads(redx)randomlyorsystematicallypileup,formingunder-collapsedsequences.Subsequentduplicationerrorsinthescaffoldingaresimilartotheheterotypeduplications.Purge_haplotigs13alignsequencestothemselvestofindasmallersequencethatalignsfullytoalargercontigorscaffold,andremovesheterotypeduplicationtypes1,3,and4.Purge_dups14additionallyusescoverageinformationtodetectheterotypeduplicationtype2andhomotypeduplications.Wedistinguishedthetwotypesofduplicationsby:1)haplotype-specificvariantsinreadsaligningathalfcoveragetoeachheterotypeduplication;2)differingconsensusqualitythatresultedfromreadcoveragefluctuationswhenaligningreadstohomotypeduplications;and3)k-mercopynumberanomaliesinwhichhomotypeduplicationswereobservedintheassemblywithmorethantheexpectednumberofcopies.ExtendedDataFig.6Falseduplicationexamplesfixedduringmanualcuration.a,Anexampleofaheterotypeduplicationinthefemalezebrafinch,non-trioassembly.Left,aself-dotplotofthisregiongeneratedwithGepard105,withsequencescolouredbyhaplotypes.Gaps,duplicatedsequences(greenandpurple),andhaplotype-specificmarkerdensitiesareindicatedatthetop.Right,adetailedalignmentviewofthegreenhaplotypeduplicationwithpaternalandmaternalmarkers,self-alignmentcomponents,transcriptsannotated,contigs,bionanomaps,andrepeatcomponentsdisplayedingEVAL95.b,Exampleofahomotypeduplicationfoundinthehummingbirdassembly.ThesewerecausedbyanalgorithmbuginFALCON,whichwaslaterfixed.c,Exampleofacombinedduplicationinvolvingbothheterotype(green)andhomotype(orange)duplications.Assemblygraphstructureisshownontheleftforclarity,highlightingtheoverlappingsitesatthecontigboundaryshadedfollowingtheduplicationtype.Assemblyerrorsincludingtheabovefalseduplicationsweredetectedandfixedduringthecurationprocess.ExtendedDataFig.7Evidenceofnear-completechromosomescaffoldsintheVGPassemblies.ShownareHi-Cinteractionheat mapsforeachspeciesaftercuration,visualizedwithPretextView106.Ascaffoldisconsideredaputativearm-to-armchromosomewhenallHi-Creadpairsinarowandcolumnmaptoasquare(thatis,anassembledchromosome)onthediagonalwithoutanyotherinteractionsoffthediagonal.Thosewithremainingoff-diagonalmatchestosmallerscaffoldsarenotlinkedbecauseofambiguousorderororientation,andareinsteadsubmittedas‘unlocalized’belongingtotherelevantchromosome.Bandsatthetopofeachheat mapshowscaffoldsidentifiedasX,Z(blue)orY,W(red)sexchromosomes.TheHi-CmapoffAstCal1isnotincludedaswehadnoremainingtissueleftoftheanimalusedtogenerateHi-Creads.ExtendedDataFig.8ComparisonofchromosomalorganizationbetweenpreviousandnewVGPassemblies.a,Zebrafinchmalecomparedtoapreviousreferenceassemblyofthesameanimal.b,Platypusmalecomparedwithapreviousreferencefemaleassembly(sotheYchromosomesareabsentinthepreviousreference).c,Hummingbirdfemalecomparedtoapreviousreferenceofthesameanimal.d,Climbingperchcomparedtoapreviousreference.EachrowrepresentsaVGP-generatedchromosomeforthetargetspecies.Coloursdepictidentitywiththereference(seekeytotheright);morethanonecolourindicatesreorganizationintheVGPassemblyrelativetothereference.Thelineswithineachblockdepictorientationrelativetothereference;apositiveslopeisthesameorientationasthereference,whereasanegativeslopeistheinverseorientation.Gapsarewhiteboxeswithnolines,inthereferencerelativetotheVGPassembly.AwhiteboxfortheentirechromosomemeansanewlyidentifiedchromosomeintheVGPassembly.Top20isthelongest20scaffoldsofthehummingbirdandclimbingperchassemblies.AccessionnumbersoftheassembliescomparedarelistedinSupplementaryTable19.ExtendedDataFig.9Haplotype-resolvedsexchromosomesandmitochondrialgenomes.a,Alignmentscatterplot,generatedwithMUMmerNUCmer107,visualizedwithdot108,ofmaternalandpaternalchromosomesfromthefemalezebrafinchtrio-basedassembly.Blue,sameorientation;red,inversion;orange,repeatsbetweenhaplotypes.ThepaternalZchromosomeishighlydivergentfromthematernalW,andthusmostlyunaligned.b,AlignmentscatterplotofassembledZandWchromosomesacrossthethreebirdspecies,approximatedwithMashMap286.Segmentsof300 kb(green),500 kb(blue),and1 Mb(purple)areshadeddarkerwithhighersequenceidentity,withaminimumof85%.ThesmallersizeandhigherrepeatcontentoftheWchromosomeareclearlyvisible.c,XandYchromosomesegmentsofthemammals(platypus,Canadalynx,palespear-nosedbat,andgreaterhorseshoebat)showingahigherdensityofrepeatswithinthemammalianXchromosomethantheavianZchromosome.d,VGPkākāpōmitochondrialgenomeassemblyrevealspreviouslymissingrepetitivesequences(adding2,232 bp)intheoriginofreplicationregion,containingan83-bprepeatunit.e,VGPclimbingperchmitochondrialgenomeassemblyshowingaduplicationoftrnL2andpartialduplicationofNad1,whichwereabsentfromthepriorreference.Orangearrowsandredlines,tRNAgenesandtheiralignments;darkgreyarrowsandgreyshading,allothergenesandtheiralignments;black,non-codingregions;greenline,conventionalstartingpointofthecircularsequence.ExtendedDataFig.10Largehaplotypeinversionswithdirectevidenceinthezebrafinchtrioassembly.a,Twoinversions(greenandred)inchromosome5foundfromtheMUMmerNUCmer107alignmentofthematernalandpaternalhaplotypeassemblies,visualizedwithdot108.b,Hi-Cinteractionplotshowingthatthetrio-binnedHi-Cdataremovemostoftheinteractionsfromtheotherhaplotype(redarrows),whichcouldbeerroneouslyclassifiedasamis-assemblyifonlyonehaplotypewasusedasareference.c,An8.5-Mbinversionfoundonchromosome11andacomplicated8.1-Mbrearrangementonchromosome13betweenmaternalandpaternalhaplotypes.d,Nomis-assemblysignalsweredetectedfromthebinnedHi-Cinteractionplots,indicatingthatthehaplotype-specificinversionsarereal.e,HalfthePacBioCLRspanandBionanoopticalmapsagreewiththeinversionbreakpointsinchromosome11,supportingthehaplotype-specificinversion.ExtendedDataFig.11Polishingartefacts.a,AnexampleofunevenmappingcoverageintheprimaryandalternatesequencepairoftheAnna’shummingbirdassembly.Inthisexample,thealternate(alt)sequencewasbuiltathigherquality,attractingalllinked-readsforpolishing.Thematchinglocusintheprimary(pri)assemblywasleftunpolished,resultinginframeshifterrorsintheTLK1gene.b,Haplotype-specificmarkers(redformaternal,blueforpaternal)anderrormarkersfoundintheassemblyontheZchromosome(inheritedfromthepaternalside)ofthetrio-binnedfemalezebrafinchassembly.Eachrowshowsmarkersbeforeshort-readpolishing,mappingallreadstobothhaplotypeassemblies,andpolishingbymappingpaternallybinnedreadstothepaternalassembly.PolishingimprovesQV,butintroduceshaplotypeswitcherrorswhenusingreadsfrombothhaplotypesasshowninrow2.Thiscanbeavoidedwhenusinghaplotypebinnedreadsforpolishing.c,Exampleofover-polishing.Thenuclearmitochondria(NuMT)sequencewastransformedasafullmitochondria(MT)sequenceduringlong-readpolishingowingtotheabsenceoftheMTcontig,wheretheNuMTattractedalllongreadsfromtheMT.Incomparison,thetrio-binnedassemblyhadtheMTsequenceassembledinplace,preventingmis-placingofMTreadsduringreadmapping.ExtendedDataFig.12Chromosomeevolutionamongthebatspeciessequenced.a,Genessurroundinganinversioninthegreaterhorseshoebat,relativetohumanchromosome15(redhighlight).TheSTARD5geneisdirectlydisruptedbythisinversion,whichseparatesexons1–5fromexon6inthegreaterhorseshoebat.b,RNA-seqtracksshowingthelackofRNAsplicingevidenceofSTARD5transcriptsinthegreaterhorseshoebat(bottom)incomparisontothepalespear-nosedbatwheretheSTARD5geneisnotdisrupted(top).c,Circosplotsofchromosomeorganizationrelationshipsbetweentheeachoftheanalysedbatsandsegmentsofthehumanchromosomes1,2,6and10.Redstar,breakpointlocationinhumanchromosome6,depictingthefissionoftheboreoeutherianchromosome5inthebatancestor;bluestar,theregionupstreamofthebreakpointinthebats;greenstar,theregiondownstreamofthebreakpointinthebats.Theredstarredbreakpointwasconfirmedasreused,asopposedtoassemblyerrors,inchromosomalrearrangementsofthepalespear-nosedbat,Egyptianfruitbat,andgreaterhorseshoebat.Thereisnoevidenceofreuseforthevelvetyfree-tailedbat.Wecouldnotconfirmbreakpointreuseinthegreatermouse-earedbatorKuhl’spipistrelleatthechromosomalscalebecausetheywereonsmallscaffoldsthatmaynotbecompletelyassembled.ExtendedDataTable1SummarymetricsofthecuratedandsubmittedvertebratespeciesassembliesFullsizetableExtendedDataTable2AnnotationsummarystatisticsinpreviousandnewlyassembledVGPreferencegenomesFullsizetableSupplementaryinformation SupplementaryInformationThisfilecontainsSupplementarytext,SupplementaryNotes1-7,SupplementaryFigures1-6andSupplementaryreferences.ReportingSummarySupplementaryTablesThisfilecontainsSupplementaryTables1-23.PeerReviewFileRightsandpermissions OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproductioninanymediumorformat,aslongasyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.Theimagesorotherthirdpartymaterialinthisarticleareincludedinthearticle’sCreativeCommonslicense,unlessindicatedotherwiseinacreditlinetothematerial.Ifmaterialisnotincludedinthearticle’sCreativeCommonslicenseandyourintendeduseisnotpermittedbystatutoryregulationorexceedsthepermitteduse,youwillneedtoobtainpermissiondirectlyfromthecopyrightholder.Toviewacopyofthislicense,visithttp://creativecommons.org/licenses/by/4.0/. ReprintsandPermissionsAboutthisarticleCitethisarticleRhie,A.,McCarthy,S.A.,Fedrigo,O.etal.Towardscompleteanderror-freegenomeassembliesofallvertebratespecies. Nature592,737–746(2021).https://doi.org/10.1038/s41586-021-03451-0DownloadcitationReceived:22May2020Accepted:12March2021Published:28April2021IssueDate:29April2021DOI:https://doi.org/10.1038/s41586-021-03451-0SharethisarticleAnyoneyousharethefollowinglinkwithwillbeabletoreadthiscontent:GetshareablelinkSorry,ashareablelinkisnotcurrentlyavailableforthisarticle.Copytoclipboard ProvidedbytheSpringerNatureSharedItcontent-sharinginitiative Furtherreading GenomicinsightsintobodysizeevolutioninCarnivorasupportPeto’sparadox XinHuang DiSun GuangYang BMCGenomics(2021) Accuratelong-readdenovoassemblyevaluationwithInspector YuChen YixinZhang ZechenChong GenomeBiology(2021) Telomere-to-telomereassemblyofafishYchromosomerevealstheoriginofayoungsexchromosomepair LingzhanXue YuGao LuohaoXu GenomeBiology(2021) LeafGo:LeaftoGenome,aquickworkflowtoproducehigh-qualitydenovoplantgenomesusinglong-readsequencingtechnology PatrickDriguez SalimBougouffa LucaErmini GenomeBiology(2021) Assemblingvertebrategenomes KatharineH.Wrighton NatureReviewsGenetics(2021) CommentsBysubmittingacommentyouagreetoabidebyourTermsandCommunityGuidelines.Ifyoufindsomethingabusiveorthatdoesnotcomplywithourtermsorguidelinespleaseflagitasinappropriate. 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