quality control analysis of the 1000 genomes project omni2 ... · quality control analysis of the...
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Quality control analysis of the 1000 Genomes Project Omni2.5 genotypes
NicoleM.Roslin1,WeiliLi1,AndrewD.Paterson1,2,3*,LisaJ.Strug1,2,41)TheCentreforAppliedGenomics,TheHospitalforSickChildren,Toronto,ON,Canada2)PrograminGeneticsandGenomeBiology,TheHospitalforSickChildren,Toronto,ON,Canada3)EpidemiologyandBiostatistics,DallaLanaSchoolofPublicHealth,UniversityofToronto,TorontoON,Canada4)BiostatisticsandStatistics,UniversityofToronto,Toronto,ON,Canada*)Correspondingauthor([email protected])
Citation Foranyuseofthe1000GenomesProjectdata,pleaseusethecitationasnotedhere:http://www.1000genomes.org/faq/how-do-i-cite-1000-genomes-project.Tocitethisreportorthelistsdescribedhere,pleaseusethefollowing:
RoslinNM,LiW,PatersonAD,StrugLJ.Qualitycontrolanalysisofthe1000GenomesProjectOmni2.5genotypes(Abstract/Program#576/F).Presentedatthe66thAnnualMeetingofTheAmericanSocietyofHumanGenetics,October18-22,2016,Vancouver,Canada.
Data Summary Chips:IlluminaHumanOmni2.5-4v1_BandIlluminaHumanOmni25M-8v1-1_BInitialnumberofSNPs:2458861Initialnumberofsamples:2318NumberofSNPspassingQC:1989184(80.9%)NumberofsamplespassingQC:2318(100%)Numberofquasi-unrelatedsampleswithconsistentethnicityandwellinferredsex:1736
Abstract The1000GenomesProjectgenotype2318individuals(48.1%male)from19populationsin5continentalgroupsontheIlluminaOmni2.5platform.Thedataarepubliclyavailable,andwillproveavaluableresourcetoobtainethnic-specificallelefrequencies,aswellasexploringpopulationhistoriesthroughprincipalcomponentsanalysis(PCA),estimationofinbreedingcoefficients,andadmixtureanalysis.Asinanystudy,thedatashouldbecleanedpriortoanalysis,toremoveindividualsormarkersofquestionablequality.Furthermore,athoroughunderstandingoftherelationshipsbetweenindividualsmustbeestablished.Herewereportourfindingsaftercomprehensiveexaminationofthedataforqualitycontrol. Thebasicqualityofthegenotypeswasassessedusingstandardprocedures.KINGversion1.4wasusedtoconfirmtherelationshipsintheprovidedpedigrees,andalsotodetectundeclaredrelationships.PCAwasusedtoexaminethesimilaritiesanddifferencesbetweenindividualsamongandbetweenpopulationgroups.
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Ingeneral,thedatawasfoundtobeofhighquality.Nosampleswereremovedduetolowcallrate(<97%)orexcessheterozygosity.Sexchromosomegenotypesshowedtwoindividualswithdiscrepanciesbetweenreportedandinferredsex,andwereunabletodeterminesexinanadditional20individuals;thesexforthesewaschangedtounknown.Relationshipcheckingfounddiscrepanciesbetweenfirst-degreerelationshipsintheprovidedpedigreesandthegenotypesin9families,includingoneinstancewhereareportedparent/childpairwasunrelated,twoinstanceswherefullsibswereunrelated,andonesetofthreeindividualswhoformedanewlydefinedtrio.Asetof1756individualswhowereinferredtobemoredistantthan3rddegreerelativeswasextractedandusedinPCA.Theseindividualsclusteredinapatternthatisconsistentwithotherpublishedreportsofglobalpopulations.Weidentified4individualswhosegenotypesclusteredmorecloselywithadifferentgeographicregionthantheoneintheprovideddata. Althoughthegenotypedataisofhighquality,errorsexistinthepubliclyavailabledatasetthatrequireattentionpriortousingthegenotypes.PLINK-formatfilesincludingSNPswithgoodqualitymetricsandrevisedpedigreestructuresisavailableathttp://tcag.ca.Fileswithdistantlyrelatedorunrelatedindividuals,withsexinferenceconsistentwithprovidedgender,andwithPCAconsistentwithcontinentalgrouparealsoavailable.
1 BackgroundGenotypesgeneratedontheIlluminaOmni2.5platformareavailablefordownloadfromthe1000GenomesProjectwebsite(http://www.1000genomes.org/).Thisdataisavaluableresourceofgenotypesfromindividualscollectedfrommultipleethnicgroupsaroundtheworldwithoutregardtodisease.Thedatacanbeusedeitherasrepresentativegenotypesfromindividualsofknownethnicityforpopulationstructureanalysis,orasasourceofethnic-specificallelefrequencieswhichcanbeusedinanalysessuchaslinkageanalysis,relationshipestimationandestimationofinbreedingcoefficients.Asinanystudy,thedatashouldbecleanedpriortoanalysis,toremoveindividualsormarkersofquestionablequality.Furthermore,athoroughunderstandingoftherelationshipsbetweenindividualsmustbeestablished.Thisreportdescribesthequalityanalysesthatweperformedonthechipdata.ListsofSNPsandsampleswithgoodqualitymetricsareavailableathttp://tcag.ca/.ThisanalysiswasapprovedbytheResearchEthicsBoardatTheHospitalforSickChildren(REBnumber1000054008).
2 DataacquisitionGenotypefiles,invcfformat,weredownloadedon16May2016fromthefollowingftpsiteatthe1000GenomesProject:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/hd_genotype_chip/(GenomesProjectetal.,2015).ThelinktothisdirectorycamefromtheFrequentlyAskedQuestionssectionofthewebsite(http://www.1000genomes.org/faq/what-omni-genotype-data-do-you-have/).GenotypesweregeneratedattheBroadandSangerInstitutes,usingtwodifferentversionsoftheOmni2.5platform.Themajorityofthesamplesweregenotypedat
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theBroad,andnoindividualsweregenotypedinbothlocations.TheanalysispresentedinthisreportstartedwiththedownloadedfileALL.chip.omni_broad_sanger_combined.20140818.snps.genotypes.vcf.gz,whichisthecombinedsetofBroadandSangergenotypes.Thedatahad2318individualsand2458861markers.SeeAppendix2forthespecificdetailsregardingthedownloadedfiles.
3 Priorprocessingby1000GenomesBeforethedatawasmadeavailablefordownload,thedataprovidersperformedasmallamountofprocessingonthedata,whichissummarizedhere.ForthedatageneratedattheBroad,genotypeswerecalledusingGenomeStudiov2010.3withthecallingalgorithm/genotypingmoduleversion1.8.4usingthedefaultclusterfile.Physicalpositionsweretakenfrombuild36,whichweresubsequentlyconvertedtohg19usingthehuman_g1k_v37dataset.SNPswereremovedaccordingtothefollowingfilters:themetadataonthechipfortheSNPwasinconsistent,theSNPhadaduplicatewithahigherGentrainScore,theassaywasdesignedformultiplealternatealleles,theSNPwasnotpolymorphicinthe1000Genomesdataset,ortheSNPwaswithin50bpofaknownindel.Thesefiltersresultedin2441898SNPsremaining(outof2450000,99.7%).ThespecificchipusedwasIlluminaHumanOmni2.5-4v1_B. ForthedatageneratedattheSangerCenter,detailedinformationonpriorprocessingwasnotprovided.However,ofthe2391739SNPsonthechip,genotypeswereprovidedfor2176330(91.0%).Notethatrsnumberswerenotprovidedforthevariantsinthisdataset.Itwasassumedthatthephysicalpositionswerebasedonhg19.ThegenotypescamefromtheIlluminaHumanOmni25M-8v1-1_Bchip. ThetwodatasetsweremergedusingGATKv3.1.1combineVariants.Thefinaldatasethad2318individualsand2458861markers.Noindividualswerepresentinbothsetsofdata.ThemajorityofSNPs(2150028,87.4%)werepresentinboththeBroadandSangerdatasets;282531(11.5%)SNPswerepresentjustinthedatageneratedattheBroad,while26302(1.1%)SNPswereonlyinthedatafromtheSanger.Inthefinaldataset,therewerenomarkerswithidenticalchromosomeandphysicalpositions.ThemergeddatawassavedtofileOmni25_sanger_broad_combined.vcf.gzandmadeavailableonthe1000Genomesftpsite.
4 FamilyandethnicinformationGenderandbasicinformationonrelationshipsbetweencertainindividualswasprovidedby1000Genomesinapedigreefile(Appendix2,file2).Additionally,reportedethnicbackgroundwasprovidedasafilefromeachgenotypingcentre(Appendix2,files3and4).Thepedigreefileincludedonlyindividualsgenotypedorsequencedinthe1000Genomesproject,andsoadditional"dummy"individualsneededtolinkuprelatedindividualswerenotpresent.Becauseofthis,severalknownrelationshipswerenotmadeexplicitbythepedigree.AllsamplesfromSangerand2098individualsfromtheBroadhadavailableethnicinformation,while
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43individualsgenotypedattheBroadhadmissingethnicityandgenderintheprovidedfiles,foratotalof2318individuals.Missinggendersandethnicitieswerefilledinusingfile5inAppendix2.CountsofindividualsfromeachofthesampledpopulationsareshowninAppendix1.
5 Qualitycontrol(QC)analysisTheprovidedvcffileswereconvertedtoPLINKbinaryformatusingPLINK1.90(https://www.cog-genomics.org/plink2/).Onemarkerhadanon-validchromosomecode(chromosomeGL000202.1forSNP11-69436716);thechromosomeforthismarkerwassettomissing.Theprovidedsexandrelationshipinformationwereincorporatedintothepedigreefiles.ThesefileswereusedasthebasisfortheQCstepsthatfollow.Analyseswereperformedusingin-housescripts,unlessotherwisestated.
5.1 BasicsamplequalityAnalyseswereperformedtodeterminethegeneralqualityofthesamples.Sincethetwodifferentgenotypingcentresusedslightlydifferentsetsofmarkers,onlythe2150028SNPspresentinboththeBroadandSangerdatasetswereusedinthesetests.
5.1.1 SexchromosomeanalysisSexchromosomecompositionwasinferredforeachofthesamplesbasedontheheterozygosityrateonchromosomeXandthecallrateonchromosomeY.ThemajorityofsamplescouldbeclearlyidentifiedasXYorXX,accordingtothefollowingcriteria:
1. XY(male)=proportionofheterozygousgenotypesontheXchromosome>0.9,andcallrateontheYchromosome>0.9.
2. XX(female)=proportionofheterozygousgenotypesontheXchromosome<0.9,andcallrateontheYchromosome<0.4.
Allotherindividualsweredeclaredtohaveambiguous(orunknown)sex.Basedonthesethresholds,twoindividuals,NA21310andHG02300,werelistedasmales,buthadgenotypesconsistentwithfemales(Figure1).Noothersexdiscrepancywasfound;however,for20individuals,thesexcouldnotbeinferredusingtheaboverules.Thesexfortheseindividualswassettomissing.For8oftheseindividuals(reportedtobemale),theheterozygosityrateontheXchromosomewasconsistentwithmales,butshowedreducedcallrateontheYchromosome.SuchalossofYcouldbetheresultofcelllineartifactsorenvironmentalfactors(Dumanskietal.,2015),ratherthangenotypingorsampleerror,andsotheseindividualswerenotflaggedashavingpoorquality.Similarly,11reportedfemalesshowedexcessivehomozygosityonchromosomeX,alsoconsistentwithcelllineartifacts.Oneindividual(HG01683:male,IBS)hadslightlyreducedcallrateonYforamale,butXchromosomeheterozygositysimilartofemales,suggestingthepossibilityofXXY.
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Figure1.ProportionofhomozygousgenotypesontheXchromosomevs.proportionofnon-missinggenotypesontheYchromosome.Symbolsarecodedbasedonthesexintheprovidedpedigreefile:male=redtriangles,female=greenplussymbols.Dashedlinesdelimitthethresholdsappliedabove.
5.1.2 CallrateTheproportionofnon-missinggenotypes(callrate)wascalculatedperindividual.Ingeneral,thecallratewasveryhigh,withanaveragecallrateof99.6%inthedata.Thelowestcallratepersamplewas97.0%,whichisatthecommonly-acceptedthresholdof97%.CallrateswerecalculatedusingPLINK1.90.
●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●
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Figure2.Proportionofnon-missinggenotypesperindividual.Thedashedlineshowsthe97%threshold.
5.1.3 MultilocusheterozygosityInordertodetectpossiblesamplecontamination,theaverageautosomalheterozygositywascalculatedforeachindividual.Theaverageheterozygositywas0.20,andnosamplewasunusuallyextreme,comparedtotheothersamples(Figure3).Thewidedistributionwithmultiplepeaksarelikelyreflectionsofthemulti-ethnicnatureofthegenotypedindividuals.
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Figure3.Histogramoftheproportionofheterozygousautosomallocipersample.
5.1.4 HeterozygoushaploidgenotypesHaploidregionsofthegenomearetypicallyrepresentedashomozygousdiploidgenotypes.TheseregionsincludetheXchromosomeformales,theYchromosome,andthemitochondrion.Individualswithanunusuallyhighnumberofheterozygousgenotypesattheselocicouldprovideadditionalevidenceofqualityissues.Sincetheregionstoexaminedependonsex,thesexinferredabovewasused,ratherthantheprovidedsex.Inthecurrentdataset,noindividualhadanunusuallyhighnumberofheterozygoushaploidgenotypes,relativetotheothersamples(resultsnotshown).ThesegenotypesweredetectedusingPLINK.
5.1.5 SummaryofbasicsamplequalityIngeneral,thequalityofthesampleswasexcellent.Therewasnoevidenceofcontamination,andnoevidenceofsamplefailure.Inferredsexwasconsistentwithprovidedsexfor2296of2318(99%)oftheindividuals.Thesexfortwoindividuals(NA21310andHG02300)waschangedfrommaletofemale,whilethesexfor20individualswassettomissing.
5.2 BasicqualityperSNPStandardtestswereperformedtodetermineSNPquality.Onlymarkersthatwerepresentonbothversionsofthechipwereincluded–theothermarkerswillhavenon-randomsetsofmissingvalues,whichwillcausebiasinmanyofthetests.Theanalyseswereperformedafterthesexchangesdescribedabove.
5.2.1 CallrateTheproportionofnon-missinggenotypeswascalculatedperSNP.Thecallratewasexcellent:2114511of2150028(98.3%)oftheSNPshadcallrate>97%(Figure4).
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Figure4.CallrateperSNP,forthemarkerspresentinboththeBroadandSangerdatasets.Thedashedlineshowsthe97%threshold.
5.2.2 Heterozygoushaploid(HH)genotypesThedistributionofheterozygoushaploidgenotypes,definedinsection5.1.4above,wasexamined.Non-missinggenotypesforfemalesontheYchromosomewerealsoidentified.Atotalof11888SNPshadatleastoneHHgenotype.NoSNPappearedtohaveanexcessivenumberoftheseproblematicgenotypes,comparedtotherestoftheSNPs(resultsnotshown).
5.2.3 Hardy-WeinbergequilibriumSNPsweretestedfordeviationfromtheproportionsexpectedunderHardy-Weinbergequilibrium(HWE),usinganexacttest(Wigginton,Cutler,&Abecasis,2005)implementedinPLINK.ForSNPsontheXchromosome,onlyinferredfemaleswereusedinthetest.SNPsontheYchromosomeandthemitochondrionwerenottested.Atotalof355357SNPs(17%)hadp-values<10-6.Althoughthiscouldindicatesubstantialproblemsintheallelecallingalgorithm,itismorelikelyduetothefactthatthesamplescamefromawidevarietyofethnicbackgrounds,andtheassumptionsofHardy-Weinbergdonothold(mostimportantlytheassumptionofrandommating)inthedata.Therefore,SNPswerenotexcludedduetodeviationfromHWE.
5.2.4 SummaryofbasicSNPqualityTheSNPshadveryhighquality.SNPswereremovedifthecallratewas<97%orifaHHgenotypewasdetected.Furthermore,SNPsthatweremonomorphicinallthesampleswerediscarded,sincetheydonotcontainanyinformation.Outoftheoriginal2150028markersthatwerepresentinboththeBroadandSangerdatasets,2105791(97.9%)passedthesequalityfilters,andwillbeusedinthemoresophisticatedanalysesbelow.
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5.3 RelationshiptestingThesoftwareKING1.4(Manichaikuletal.,2010)wasusedtoinfercloserelationshipsbetweenpairsofsamples.Thisprogramestimateskinshipcoefficientsforallpairsofindividualsbasedontheirheterozygosity,anddoesnotrequireallelefrequencyestimates.Thehighqualitysetof2105791SNPsdescribedabovewasused.KINGwasalsousedtoselectasetofindividualswhowereinferredtobeunrelatedormoredistantlyrelatedthan3rddegree(firstcousinorequivalent). Priortotesting,therelationshipinformationprovidedby1000Genomeswasincorporated.Therewere28pedigreesthatwerelargerthantrios,showninAppendix3,plusanadditional373trios(basedonthenumberofindividualsperfamilyID).Inmanycases,largerfamilieswerecomposedofmultiplesmallerfamilieswithdifferentpedigreeIDs.WeassignedtheseanewfamilyIDbasedonthepopulationfromwhichthefamiliescame.Forexample,thethreeindividualsHG00501(amotherinatrio,familySH028),HG00512(afatherinatrio,familySH032)andHG00524(afatherinatrio,familySH036)werealllistedassiblings,eventhoughtheyhaddifferentfamilyIDs.Wecombinedalltheetriosintoasinglefamily,addingdummyparents,andnamedtheextendedfamilyCHS3. Withinthe28pedigrees,thepairwisekinshipcoefficientswereconsistentwiththeprovidedstructurefor23ofthem.Fortheremaining5families,ASW3wassplitintotwoseparatefamilies(theremayhavebeenatypointheprovidedpedigreeinformation,mixingupIDsNA20334andNA20344).FamiliesASW4andASW5werecombinedandonehalfsiblingchangedfathers.FamilyLWK003originallyconsistedoftwoindividualswithacommonmother,withnoinformationonthefather(s).Thegenotypeswereconsistentwithafullsiblingrelationship,andsoasingledummyfatherwascreated.Finally,familyYRI4wassplitintotwounrelatedfamilies. InthetrioCLM23,thekinshipcoefficientbetweentheparentswasestimatedtobe0.033,indicatingthattheymaybedistantlyrelated(consistentwithfirstcousinonceremoved,orequivalentrelationship).Sincetherearenootherfamilymemberswithgenotypesavailable,itisnotpossibletoconfirmthisrelationship,andsothepedigreewasnotchanged.Apaperestimatedinbreedingcoefficientsin1000Genomessamplesbasedonwholegenomesequencedata(Gazal,Sahbatou,Babron,Genin,&Leutenegger,2015),includingtheparentsoftrioCLM23.Thefatherofthetrio(HG01277)wasfoundtohaveaninbreedingcoefficientestimateconsistentwithoffspringofsecondcousins.However,thechildinthetrio(HG01279)wasnotanalyzed,andsothisanalysiscannotconfirmarelationshipbetweentheparents. Otherthanthechangesmadetotheextendedfamilies,5additionalchangesweremade,basedoninferredfirst-degreerelationshipsbetweenpairsofindividualswithdifferentfamilyIDs.Therewere3instancesofunreportedfullsiblingrelationships,oneinstancewherethreesupposedlyunrelatedindividualsformedatrio,andoneinstancewhereareportedparent/offspringrelationshipwasinferredtobeunrelated.Asubstantialnumberof2nddegreerelationshipswerealsoobserved,butpedigreeswerenotmodifiedbasedonthesemoredistantkinshipcoefficients.AllpedigreechangesareshowninAppendix4.
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KINGwasusedtoselectquasi-unrelatedindividuals.Itidentified1756individualswhowerenocloserthan3rddegreerelatives(firstcousinorequivalent).
5.3.1 MendelianerrorsTheprogramPEDSTATS0.6.10(Wigginton&Abecasis,2005)wasusedtolookforerrorsinMendeliantransmissionintherevisedpedigreesidentifiedabove.OnlytheautosomesandchromosomeXwereexamined.Atotalof228542errorswerefound.Outofthepedigreeshavingatleastoneparent/childpair,family1349hadthemosterrors(5743).Sinceover2millionSNPsweretested,thisrepresentserrorsinapproximately0.2%ofthemarkers,andsodoesnotpresentaqualityconcern.ThedistributionoferrorsbySNPwasalsonotremarkable(resultsnotshown).All116607SNPswith≥1errorwereremoved,resultingin1989184SNPs.
5.4 PrincipalcomponentsanalysisPrincipalcomponentsanalysis(PCA)wasusedasavisualaidtoshowhowsimilarthegenotypesfromthedatawere,whentheprovidedethnicinformationwasincluded.Inordertoeasecomputationalburden,andtoavoidartifactsfromunusualpatternsoflinkagedisequilibrium(LD)thatareunrelatedtoethnicity,thesetof1989184SNPswasfurtherreduced.First,allmarkersonchromosomeXwereremoved.Next,tworegionswithunusualpatternsoflinkagedisequilibrium(LD)wereremoved:themajorhistocompatibilityregiononchromosome6,andaninversionpolymorphismonchromosome8.PositionsonthegeneticmapwereobtainedfromtheRutgerscombinedmap(Matiseetal.,2007),andonlymarkerswithuniquecMpositions(upto3decimalplaces)wereretained.Arbitrarily,outofagroupofSNPsatthesamegeneticposition,theonewiththelowestphysicalpositionwasretained.Markerswithminorallelefrequency(MAF)>0.3inthecompletesetofdatawereextracted.Thisresultedinasetof282273markers.ToreducetightLDbetweenmarkers,thesetofSNPswasfurtherreducedsothatallmarkershadpairwiser2<0.2withineachchromosome,toproduceafinalsetof57931SNPs.SincecloserelationshipsbetweenindividualscandistortthePCAresults,onlythe1756quasi-unrelatedindividualsidentifiedabovewereusedintheanalysis.PCAwasperformedonthisdatausingtheSmartPCApackage(version10210)ofEigenstrat(Patterson,Price,&Reich,2006;Priceetal.,2006).
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Althoughonlyasubsetofindividualsrecruitedforthe1000GenomesProjecthaveOmni2.5genotypes,individualsfromallcontinentalgroupsarerepresented.ThedistributionofsamplesinthePCAisconsistentwithwhathasbeenshownelsewhere,withAFR,EASandEURformingmajoraxes,andAMRandSASlyingwithintheseaxes(Figure5).Fourindividualsdidnotclusterwellwithotherindividualsfromtheircontinentalgroups.ThreeindividualsfromAMR(HG01241,HG01242andHG01108,allfromPUR)clusteredmorecloselywithindividualsfromAFR,andoneindividualfromAFR(NA20314,ASW)wasmoresimilartoAMR.Theseresultsarenotsurprising,giventhepopulationhistories.Thetwoindividualswithsexdiscrepancyclusteredwellwithintheirlistedgeographicregion.
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−0.03 −0.01 0.01 0.03
−0.02
0.02
0.06
0.10
PC2
PC3
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−0.04 −0.02 0.00 0.02
−0.03
−0.01
0.01
0.03
PC1
PC2
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−0.04 −0.02 0.00 0.02
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0.02
0.06
0.10
PC1
PC3
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Figure5.Threedimensionalplotofthefirstthreeprincipalcomponents(PC1,PC2,PC3)of1756quasi-unrelatedindividuals.Pointsareshadedbytheirpopulationcodes.Ingeneral,green=AFR,blue=AMR,yellow=EAS,red=EUR,andorange=SAS.
30September2016 11
The4individualswhodidnotclusterwellwiththeirgeographicregionwereremoved,andclusteringwasrepeatedwithineachgeographicregion.ForAFR,LWK,MKKandYRIeachformedtightclusters,whileACBandASWweremorespreadout(Figure6).
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Figure6.PlotsofthefirsttwoPCsfromananalysisofAFR.Eachplothighlightsonepopulationfromthiscontinentalgroup.
30September2016 12
InAMR,the4differentpopulationsdidnotformtightclusters(Figure7).Also,eventhoughtheycouldbedifferentiatedsomewhat,theyalsoshowedsubstantialamountsofoverlap.
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Figure7.PlotsofthefirsttwoPCsfromananalysisofAMR.Eachsub-populationishighlightedinaseparateplot.
30September2016 13
Amongthe5EASpopulations,JPTwasdistinctfromCDX,CHB,CHSandKHV(Figure8).OneJPTsamplemaybeadmixedwithanancestorfromaChinesepopulation.CDX(ChineseDai)andKHV(KinhfromVietnam)weremoresimilartoeachotherthantotheotherpopulationsfromChina,withtheexceptionofoneCDXindividualwhoclusteredwitheitherCHBorCHS.
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Figure8.PlotsofthefirsttwoPCsfromananalysisofEAS.Eachsub-populationishighlightedinaseparateplot.
30September2016 14
The5populationsfromEURformedthreemainclusters(Figure9).CEUandGBRwereindistinguishablefromeachother,whileIBSandTSIshowedsimilaritiestoeachother.FINwasdistinctfromtheother4populations.
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PC2
Figure9.PlotsofthefirsttwoPCsfromananalysisofEUR.Eachsub-populationishighlightedinaseparateplot.
SinceonlyoneethnicgroupfromSASwasincludedintheOmni2.5genotyping(GIH),PCAwasnotperformedwithinthiscontinentalgroup. Intotal,therewere1752individualswhowereunrelated(upto3rddegree),andwhoclusteredwellwithintheirassignedgeographicregion.
6 SummaryTheOmni2.5genotypesprovidedbythe1000GenomesProjectwereanalyzedforquality.Ingeneral,thequalityofthedatawasexcellent.Outof2318samples,twoindividualshadgenotypesontheXandYchromosomesthatwereinconsistentwiththeprovidedgender,whilesexcouldnotbedeterminedforanother20individuals.Itisrecommendedthatthese22individualsberemovedforanyanalysisinwhichsexisimportant.Allsampleshadcallrate>97%.Nosamplehadunusuallyhighheterozygosityacrosstheautosomes.Over98%oftheSNPshadcallrate>97%. Dummyindividualswereaddedtotheprovidedfamiliestoform"complete"pedigrees.Basedonpairwiserelationshiptesting,thepedigreesweremodifiedtobeconsistentwithfirst-degreeestimatedkinshipcoefficients.Changesweremadetoatotalof10pedigrees. Asetofunrelatedordistantlyrelatedindividualswasalsochosen.Theseindividualsweremoredistantthan3rddegreerelatives,clusteredwithother
30September2016 15
samplesfromtheircontinentalgroup,andhadclearsexinferencethatwasconsistentwiththeprovidedgender.Thissethad1736individuals. Asetof1989184highqualitySNPswasselected.ThesemarkerswerepresentinthedatasetsfromboththeBroadandSangerInstitutes,hadcallrate>97%,hadtwoobservedalleles,andnoobservederrorsinMendeliantransmission. Thefollowingfilesareavailableatourwebsite(http://tcag.ca):
• PLINKbinaryformatfilesincludingthe1989184SNPspassingQCwithtwoalleles,andtheinferredpedigreestructures,includingallnecessarydummyindividuals
• PLINKbinaryformatfilesincludingthe1989184SNPspassingQCwithtwoalleles,andthe1736individualswhowereinferredtobe<3rddegreerelatives,clusteredwellwithotherindividualsintheirgeographicregion,andhadclearsexinferencethatwasconsistentwithprovidedgender
• Tableofqualitystatisticspersample• TableofqualitystatisticsperSNP
30September2016 16
Appendix 1:Listof1000Genomespopulationsanddescriptions(numberofindividualswithOmni2.5data)AFR(Africans,508)ACB=AfricanCaribbeaninBarbados(102)ASW=AfricanancestryinSouthwestUS(104)LWK=LuhyainWebuye,Kenya(116)MKK=MaasaiinKinyawa,Kenya(31)YRI=YorubainIbadan,Nigeria(189)AMR(Americas,418)CLM=ColombianinMedellin,Colombia(107)MXL=MexicanancestryinLosAngeles,California(103)PEL=PeruvianinLima,Peru(105)PUR=PuertoRicaninPuertoRico(111)EAS(EastAsians,587)CDX=ChineseDaiinXishuangbanna,China(100)CHB=HanChineseinBejing,China(108)CHD=ChineseinDenver,Colorado(1)CHS=SouthernHanChinese,China(153)JPT=JapaneseinTokyo,Japan(105)KHV=KinhinHoChiMinhCity,Vietnam(121)EUR(Europeans,649)CEU=UtahresidentswithNorthernandWesternEuropeanancestry(183)FIN=FinnishinFinland(100)GBR=BritishinEnglandandScotland(104)IBS=IberianpopulationsinSpain(150)TSI=ToscaniinItaly(112)SAS(SouthernAsians,113)GIH=GujaratiIndianinHouston,TX(113)
30September2016 17
Appendix 2:Listofdownloadedfiles1. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/h
d_genotype_chip/ALL.chip.omni_broad_sanger_combined.20140818.snps.genotypes.vcf.gz(lastmodifiedAugust182014;mergedgenotypefile,invcfformat)
2. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/working/20140502_sample_summary_info/20140502_all_samples.ped(lastmodifiedMay2,2014;sexandrelationshipinformation)
3. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/hd_genotype_chip/broad_intensities/omni25.2141.sample.panel(lastmodifiedNovember222013;populationinformationforsamplesgenotypedattheBroad)
4. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/hd_genotype_chip/sanger_intensities/sanger_omni_chip.20130805.ALL.panel(lastmodifiedAugust122013;populationinformationforsamplesgenotypedatSanger)
5. http://www.1000genomes.org/sites/1000genomes.org/files/documents/20101214_1000genomes_samples.xls(populationinformationfor43individualsmissingfromBroadsamplespopulationfile)
30September2016 18
Appendix 3:Pedigreeslargerthantriosbasedontheinformationprovidedby1000Genomes;originalpedigreeidentifiersareshowninnon-blackcolours
Family ASW3
ASW34NA20344ASW33NA20336ASW35 NA20349ASW36NA20334
ASW32ASW31
NA20345 NA20350NA20337NA20335
2484 2485 2489 2492
Family ASW1
ASW13NA19713NA19982 NA19985
ASW11 ASW12
NA19714NA19983
2436
2437
Family ASW2
NA20341NA20289ASW23
ASW22ASW21
NA20290
2471
2487
Note:family2487hasanothermember,NA20340,whoisunrelatedtoeveryoneinASW2
Family ASW4
NA20301ASW44 NA20282 NA20278 ASW43
ASW42ASW41
NA20302NA20285 NA20279NA20284
24692467
2477
Family ASW5
NA20312 NA20313
ASW51 ASW52
2478i 2478ii
30September2016 19
(Appendix 3, continued)
Family CDX4
HG02381HG02373
CDX42CDX41
HG02373
HG02381
Family CDX3
HG00983HG00978
CDX32CDX31
HG00983HG00978
Family CEU1
NA06986 NA12813 NA12812NA07045
CEU12 CEU11
NA12801NA06997
13291 1454
Family CHS1
HG00580 HG00635HG00581HG00583 HG00578HG00577 HG00634HG00584
CHS11 CHS12 CHS14CHS13
HG00636HG00582HG00579HG00585
SH054 SH052 SH053 SH071
Note:familiesSH058andSH062aremoredistantlyrelatedtofamilyCHS1
Family CHS2
HG00701 HG00702HG00658
HG00657HG00656
HG00703
SH089
SH074
Family CHS3
HG00525HG00500 HG00512 HG00524HG00513HG00501
CHS31 CHS32
HG00526HG00514HG00502
SH028 SH032 SH036
30September2016 20
(Appendix 3, continued)
Family GBR002
HG00147 HG00146
GBR0022 GBR0021
GBR002aGBR002
Family GIH004
NA20874 NA20879
GIH0041 GIH0042
NA20879NA20874
Family KHV1
HG02046 HG02067
KHV11 KHV12
VN063VN056
Family LWK003
NA19444NA19434
LWK0031 LWK0032NA19432
Family LWK005
LWK0053 NA19470NA19443
LWK0052LWK0051
NA19469LWK005
NA19443
Family LWK007
NA19396 NA19397
LWK0071 LWK0072
NA19397NA19396
Family LWK008
NA19373 NA19374
LWK0081 LWK0082
NA19373 NA19374
Family LWK009
NA19347 NA19352
LWK0091 LWK0092
NA19347NA19352
30September2016 21
(Appendix 3, continued)
Family MXL1
NA19676 NA19680NA19675
NA19679 NA19678
NA19677 m004
m009
Family MXL2
NA19672NA19661 MXL23NA19660
NA19686
MXL21 MXL22
NA19662 NA19674NA19684NA19685
m008
m011
2382
Family PEL100
HG02299 HG02302 HG02301HG02298
PEL1001 PEL1002
HG02303HG02300
PEL52 PEL53
HG02300showssexinferredbygtypes(maleinpedfile)
Family TSI1
NA20792NA20526
TSI12TSI11
NA20526NA20792
Family YRI1
NA18913 NA18912NA19240
NA19239 YRI11NA19238
NA18914
Y117
Y028
30September2016 22
(Appendix 3, continued)
Family YRI2
NA18861NA18862
YRI21 NA19105
NA18863
Y024
Y080
Family YRI3
NA19152 NA19104NA19153
YRI31YRI32
NA19154
Y072Y080
Family YRI4
NA19185 NA19166 NA19204NA19203NA19184
YRI42 YRI41
NA19205NA19186
Y039Y049 Y048Family YRI5
NA19214 NA19254NA19213
YRI51YRI52
NA19215
Y062Y110
30September2016 23
Appendix 4:PedigreesthatweremodifiedafterrelationshipinferenceinKING.Pedigreespriortotestingareshowninblueboxes,whilepedigreesaftertestingareshowninredboxes.
Family ACB1
HG02478 HG02479HG02429
ACB12ACB11
HG02480
Family BB53
HG02480
HG02478 HG02479
HG02429
FamilyBB39
HG02429andHG02478inferredtobefullsibs
Family ASW3
ASW35 ASW36 NA20336NA20334
ASW31 ASW32
NA20337NA20335
Family ASW6
ASW63 NA20349 ASW64NA20344
ASW61 ASW62
NA20350NA20345
ASW3splitintotwodisjointfamilies
Family ASW3
ASW34NA20344ASW33NA20336ASW35 NA20349ASW36NA20334
ASW32ASW31
NA20345 NA20350NA20337NA20335
30September2016 24
(Appendix 4, continued)
Family ASW4
NA20301ASW44 NA20282 NA20278 ASW43
ASW42ASW41
NA20302NA20285 NA20279NA20284
Family ASW5
NA20312 NA20313
ASW51 ASW52
Family ASW4
NA20282 NA20301ASW44 ASW45ASW43NA20278
ASW42ASW41
NA20302NA20284 NA20313NA20279 NA20285
NA20312
ASW4andASW5combined,withoneunrelatedindividualsplitoff;halfsiblingsswitched
30September2016 25
(Appendix 4, continued)
Family ASW7
NA20274 NA20414
ASW71 ASW72
Family GIH005
NA20900
NA20891 NA20882
NA20274
NA20414
NA20882
NA20900
NA20891
twoindividualsinferredtobefullsiblings
threeindividualsinferredtobeatrio
Family LWK001
NA19313
NA19331 LWK0011
NA19334
Family LWK001
LWK0011 NA19334NA19331
LWK0013LWK0012
NA19313
twoindividualsinferredtobefullsiblings
30September2016 26
(Appendix 4, continued)
Family 2395
NA19742
23951 NA19740
Family LWK003
NA19434 NA19444
LWK0031 NA19432
Family 2395
NA19742
NA19741 NA19740
NA19741
Family LWK003
NA19444NA19434
LWK0031 LWK0032NA19432
siblingsofunknowntypeinferredtobefullsibs
parent/offspringinferredtobeunrelated
Family YRI4
NA19185 NA19166 NA19204NA19203NA19184
YRI42 YRI41
NA19205NA19186
Family YRI4
NA19166 NA19185NA19184
YRI41 YRI42
NA19186
Family Y048
NA19205
NA19203 NA19204
YRI4splitintotwodisjointfamilies
30September2016 27
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