Title:ProfilingadaptiveimmunerepertoiresacrossmultiplehumantissuesbyRNASequencing

Authors:SergheiMangul*#1,2,IgorMandric*3,HarryTaegyunYang1,NicolasStrauli4,DennisMontoya5,JeremyRotman1,WillVanDerWey1,JiemR.Ronas6,BenjaminStatz1,DouglasYao5,AlexZelikovsky3,RobertoSpreafico2,SagivShifman**7,NoahZaitlen**8,MauraRossetti**9,K.MarkAnsel**10,EleazarEskin**#1,11

Affiliations:

1DepartmentofComputerScience,UniversityofCaliforniaLosAngeles,LosAngeles,CA,USA

2InstituteforQuantitativeandComputationalBiosciences,UniversityofCaliforniaLosAngeles,LosAngeles,CA,USA

3DepartmentofComputerScience,GeorgiaStateUniversity,Atlanta,USA

4BiomedicalSciencesGraduateProgram,UniversityofCalifornia,SanFrancisco,CA,USA

5DepartmentofMolecular,Cell,andDevelopmentalBiology,UniversityofCalifornia,LosAngeles,LosAngeles,CA,

USA

6Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los

Angeles,CA90095,USA

7DepartmentofGenetics,TheInstituteofLifeSciences,TheHebrewUniversityofJerusalem,Jerusalem,Israel

8DepartmentofMedicine,UniversityofCalifornia,SanFrancisco,CA,USA

9Immunogenetics Center,Department of Pathology and LaboratoryMedicine,DavidGeffen School ofMedicine,

UniversityofCaliforniaLosAngeles,LosAngeles,CA,USA

10DepartmentofMicrobiologyandImmunology,SandlerAsthmaBasicResearchCenter,UniversityofCalifornia,San

Francisco,SanFrancisco,CA,USA

11DepartmentofHumanGenetics,UniversityofCaliforniaLosAngeles,LosAngeles,USA

#Correspondenceto:SergheiMangulsmangul@ucla.eduandEleazarEskineeskin@cs.ucla.edu

*Equalcontribution

**Equalcontribution

Abstract

Assay-basedapproachesprovideadetailedviewoftheadaptiveimmunesystembyprofilingT

andBcellreceptorrepertoires.However,thesemethodscarryahighcostandlackthescaleof

standard RNA sequencing (RNA-Seq). Here we report the development of ImReP, a novel

computationalmethodforrapidandaccurateprofilingoftheadaptiveimmunerepertoirefrom

regularRNA-Seqdata.Weappliedournovelmethod to8,555samplesacross544 individuals

from 53 tissues from the Genotype-Tissue Expression (GTEx v6) project. ImReP is able to

efficientlyextractTCR-andBCR-derived reads fromRNA-Seqdata. ImRePcanalsoaccurately

assemblethecomplementarydeterminingregions3(CDR3s),themostvariableregionsofBand

T cell receptors, and determine their antigen specificity. Using ImReP, we have created a

systematicatlasofimmunologicalsequencesforBandTcellrepertoiresacrossabroadrangeof

tissuetypes,mostofwhichhavenotbeenstudiedforBandTcellreceptorrepertoires.Wealso

compared the GTEx tissues to track the flow of T- and B-clonotypes across immune-related

tissues,includingsecondarylymphoidorgansandorgansencompassingmucosal,exocrine,and

endocrinesites,andweexaminedthecompositionalsimilaritiesofclonalpopulationsbetween

these tissues. The atlas of T and B cell receptors, freely available at

https://sergheimangul.wordpress.com/atlas-immune-repertoires/, is the largest collection of

CDR3sequencesandtissuetypes.Weanticipatethisrecoursewillenhancefutureimmunology

studiesandadvancedevelopmentoftherapiesforhumandiseases.ImRePisfreelyavailableat

https://sergheimangul.wordpress.com/imrep/.

Introduction

Akeyfunctionoftheadaptiveimmunesystem,whichiscomposedofB-cellsandT-cells, isto

mountprotectivememoryresponses toagivenantigen.BandTcells recognizetheirspecific

antigens through their surface antigen receptors (B and T cell receptors, BCR and TCR,

respectively),whichareuniquetoeachcellanditsprogeny.BCRandTCRarediversifiedthrough

somaticrecombination,aprocessthatrandomlycombinesvariable(V),diversity(D),andjoining

(J)genesegments,andinsertsordeletesnon-templatedbasesattherecombinationjunctions1

(Figure1a).TheresultingDNAsequencesarethentranslatedintotheantigenreceptorproteins.

Thisprocessallowsforanastonishingdiversityofthelymphocyterepertoire(i.e.,thecollection

of antigen receptors of a given individual), with >1013 theoretically possible distinct

immunological receptors1. This diversity is key for the immune system to confer protection

againstawidevarietyofpotentialpathogens2.Inaddition,uponactivationofaB-cell,somatic

hypermutationfurtherdiversifiesBCRsintheirvariableregion.Thesechangesaremostlysingle-

basesubstitutionsoccurringatextremelyhighrates (10–5 to10–3mutationsperbasepairper

generation)3. Isotype switching is another mechanism that contributes to B-cell functional

diversity.Here,antigenspecificityremainsunchangedwhiletheheavychainVDJregionsjoinwith

different constant (C) regions, such as IgG, IgA, or IgE isotypes, and alter the immunological

propertiesofaBCR.

High-throughputtechnologiesenableunprecedentedaccuracywhenprofilingtheBCRandTCR

repertoires.Commonlyusedassay-basedapproachesprovideadetailedviewof theadaptive

immunesystemwithdeepsequencingofamplifiedDNAorRNAfromthevariableregionofBCR

orTCRloci(Rep-Seq)4.Thosetechnologiesareusuallyrestrictedtoonechain,withthemajority

of studies focusing on the beta chain of TCRs and the heavy chain of BCRs. Recent studies2

successfully applied assay-based approaches to characterize the immune repertoire of the

peripheralblood.However,littleisknownabouttheimmunologicalrepertoiresofotherhuman

tissues,includingbarriertissueslikeskinandmucosae.Studiesinvolvingassay-basedprotocols

usually have small sample sizes, thus limiting analysis of intra-individual variation of

immunologicalreceptorsacrossdiversehumantissues.

RNASequencing(RNA-Seq)traditionallyusesthereadsmappedontohumangenomereferences

to study the transcriptional landscape of both single cells and entire cellular populations. In

contrasttoassay-basedprotocolsthatproducereadsfromtheamplifiedvariableregionofBCR

orTCRloci,RNA-Seqisabletocapturetheentirecellularpopulationofthesample,includingB

andTcells.However,duetotherepetitivenatureoflociencodingforBCRsandTCRs,aswellas

theextremelevelofdiversityinBCRandTCRtranscripts,mostmappingtoolsareillequippedto

handle immune repertoire sequences. Despite this, BCR and TCR transcripts often occur in

sufficient numberswithin the transcriptome ofmany tissues to characterize their respective

immunologicalrepertoires5.

In this study, we developed ImReP, a novel computational method for rapid and accurate

profilingoftheadaptiveimmunerepertoirefromregularRNA-Seqdata.Weapplieditto8,555

samplesacross544individualsfrom53tissuesobtainedfromGenotype-TissueExpressionstudy

(GTExv6)6.Thedatawasderivedfrom38solidorgantissues,11brainsubregions,wholeblood,

andthreecell lines. ImReP isabletoefficientlyextractTCR-andBCR-derivedreadsfromthe

RNA-Seq data and accurately assemble the complementarity determining regions 3 (CDR3s).

CDR3 are the most variable regions of B and T cell receptors and determine the antigen

specificity.UsingImReP,wecreatedasystematicatlasofimmunologicalsequencesforB-andT-

cellrepertoriesacrossabroadrangeoftissuetypes,mostofwhichwerenotpreviouslystudied

for B- and T-cell repertoires. We also examined the compositional similarities of clonal

populationsbetweenthetissuestotracktheflowofTandBclonotypesacrossimmune-related

tissues, including secondary lymphoid and organs that encompass mucosal, exocrine, and

endocrinesites.OurproposedapproachisnotsuperiorincomparisontotargetedTCRorBCR;

rather, itprovidesauseful tool formining large-scaleRNA-Seqdatasets forstudyofadaptive

immunerepertoires.

Results

ImReP:atwostageapproachforadaptiveimmunerepertoiresreconstruction

WeappliedImRePto0.6trillionRNA-Seqreads(92Tbp)from8,555samplestoassembleCDR3

sequencesofBandTcellreceptors(TableS1).TheRNA-SeqdatawasgeneratedbytheGenotype-

Tissue Expression Consortium (GTEx v6). First, we mapped RNA-Seq reads to the human

referencegenomeusingashort-readaligner(performedbyGTExconsortium6)(Figure1).Next,

weidentifyreadsspanningtheV(D)JjunctionofBandTcellreceptorsandassembleclonotypes

(agroupofcloneswithidenticalCDR3aminoacidsequences).HereImRePused0.02trillionhigh

qualityreadsthatsuccessfullymappedtoBCRgenes,successfullymappedtoTCRgenes,orwere

unmappedreadsthatfailedtomaptothehumanreferencegenome(Figures1aandS1).

ImReP is a two-stageapproach toassembleCDR3 sequencesanddetect correspondingV(D)J

recombinations(Figure1b).Inthefirststage,ImRePutilizesreadsthatsimultaneouslyoverlapV

andJgenesegmentstoinfertheCDR3sequences.WedefinetheCDR3asthesequenceofamino

acidsbetweenthecysteineontherightofthejunctionandphenylalanine(forallTCRchainsand

immunoglobulinlightchains)ortryptophan(forIGH)ontheleftofthejunction.Inthesecond

stage, ImReP utilizes reads that overlap a single gene segment containing a partial CDR3

sequence.ImRePthenusesasuffixtreetoperformpairwisecomparisonofthereadsandjoin

thereadsbasedonoverlapintheCDR3region.Further,ImRePusesaCASTclusteringtechnique7

toaccuratelyassembleclonotypes forPCRandsequencingerrors.WemapDgenes (for IGH,

TCRB,andTCRG)ontoassembledCDR3sequencesandinfercorrespondingV(D)Jrecombination.

AdetaileddescriptionofthemethodologyimplementedwithImRePisprovidedintheExtended

Experimental Procedures Section. ImReP is freely available at

https://sergheimangul.wordpress.com/imrep/.

To validate the feasibility of using RNA-Seq to study the adaptive immune repertoire, we

simulatedRNA-SeqdataasamixtureoftranscriptomicreadsandreadsderivedfromBCRand

TCR transcripts (Figure S3). BCR and TCR transcripts are simulated based on random

recombinationofVand Jgenesegments (obtained from IMGTdatabase8)withnon-template

insertionattherecombinationjunction(FigureS2).WeassessedtheabilityofImRePtoextract

CDR3-derived reads from the RNA-Seq mixture by applying ImReP to a simulated RNA-Seq

mixture.Whileoursimulationapproachmaynotcompletelysummarizethevariousnuancesand

eccentricitiesofactualimmunerepertoires,itallowsustoassesstheaccuracyofourtool.ImReP

is able to identify 99% of CDR3-derived reads from the RNA-Seq mixture, suggesting it is a

powerful tool for profiling RNA-Seq samples of immune-related tissues. Details about the

simulationdataareprovidedintheExtendedExperimentalProceduressection.

Next,wecomparedImRePwithothermethodsdesignedtoassembleimmunerepertoires.We

alsoinvestigatedthesequencingdepthandreadlengthrequiredtoreliablyassembleTCRand

BCR sequences from RNA-Seq data. Our simulations suggest that both read length and

sequencingdepthhaveamajor impactonprecision-recall ratesofCDR3 sequenceassembly.

ImRePisabletomaintainan80%precisionrateforthemajorityofsimulatedscenarios.Average

CDR3coveragethatishigherthan8allowsImRePtoarchivearecallratecloseto90%foraread

length above 75bp (Figure 2a). Increasing coverage has a positive effect on the number of

assembledclonotypesachievedbyImRePforbothBandTcellreceptors.Ingeneral,weobserve

higherprecision-recallratesofCDR3sequenceassemblyforTCRsincomparisontoBCRs(Figure

2a-b).

We compared the performance of ImReP to MiXCR (RNA-Seq mode)9, TRUST10, TraCeR11,

V’DJer12, IgBlast-basedpipeline13, and iSSAKE14. These toolsweredeveloped to assemble the

hypervariablesequencesintheTandBcellreceptorsdirectlyfromRNA-Seqdata.Wesupplied

eachofthosetoolswiththeoriginalRNA-Seqreadsasrawormappedreads,dependingonthe

softwaredevelopers’recommendations.).TRUSTandTraCeRdonotsupporttheanalysisofBCR

sequencesandwereexcludedfromthecomparisonbasedfortheIGHdata.iSSAKEisnolonger

supportedandwasnotrecommendedforuse.Unfortunately,weobtainedemptyoutputafter

running V’DJer, and increasing coverage in the simulated data did not solve the problem.

Alternativeapproaches,suchasIMSEQ15,cannotbeapplieddirectlytoRNA-Seqreadsbecause

theywere originally designed for targeted sequencing of B or T cell receptor loci. Thus, to

independentlyassessandcompareaccuracywithImReP,weonlyranIMSEQwiththesimulated

readsderivedfromBCRorTCRtranscripts(FigureS1).Scriptsandcommandstorunalltoolsused

inthisstudyareprovidedintheExtendedExperimentalProceduresandareavailableonlineat

https://github.com/smangul1/Profiling-adaptive-immune-repertoires-across-multiple-human-

tissues-by-RNA-Sequencing.ImRePconsistentlyoutperformedexistingmethodsonIGHdatain

bothrecallandprecisionratesforthemajorityofsimulatedparameters.ImRePandMiXCRshow

similarperformanceonTCRAdataandoutperformothermethods.Notably,ImRePwastheonly

methodwithacceptableperformanceon IGHdataat50bpread length,reconstructingwitha

higherprecisionratesignificantlymoreCDR3clonotypesthanothermethods.

Todemonstrate the feasibilityof applyingnon-specificRNASequencing toassemble immune

repertoiresequences,weusedtheTCRB-Seqdatapreparedfromthreesamplesofkidneyrenal

clearcellcarcinoma(KIRC)byLi,Bo,etal.10.WedownloadedmatchingRNA-Seqsamplesfrom

theTCGAportal.Intotal,weobtained301million2x50bpreadsfromthreeRNA-Seqsamples.

First,wepreparedtheCDR3sequencesobtainedfromTCRB-Seqandconsideredonlycomplete

CDR3s,whichwedefinedasasequenceofaminoacidsstartingwithcysteine(C)andendingwith

phenylalanine (F).We considered theprepared, completeCDR3sobtained fromTCRB-Seq as

totalimmunerepertoire.

WeusedImReP,MIXCR,TRUST,andIMSEQtoassembleCDR3softheTCRBchain.Weexcluded

V’DJer,becauseitonlysupportsimmunoglobulinchainsandisnotsuitabletoassembleCDR3s

fromTcellreceptors.ImReP,MIXCR,andTRUSTassembledcomparablenumbersofcomplete

CDR3sthatfullymatchCDR3sfromthetotalimmunerepertoireobtainedbyTCRB-Seq;IMSEQ

assemblednoneoftheCDR3sequences(Figure2candFigureS4).Onaverage,54%ofCDR3s

assembledby ImRePfullymatchCDR3sfromTCRB-Seq.Ourmethodwasabletorecover0.1-

0.9%ofthetotalimmunerepertoireobtainedbyTCRB-Seqfromkidneytissue.Othertissues,

including spleen andwhole blood, contain a higher fraction of T cells and allow RNA-Seq to

capture a higher fraction of the total immune repertoire. One should note, the number of

completeCDR3sfullymatchingCDR3sobtainedbyTCRB-Seqinourstudy(reportedinFigure2c

andFigureS4)arenotfullycomparablewiththeresultsreportedinLi,Bo,etal.10,whereCDR3

sequencesareconsideredtomatchCDR3sfromTCRB-Seqifatleast6aminoacidsarematched.

Scripts and commands utilized to process the data and run repertoire assembly tools are

provided in Extended Experimental Procedures and are available online at

https://github.com/smangul1/Profiling-adaptive-immune-repertoires-across-multiple-human-

tissues-by-RNA-Sequencing.

WefurthervalidatedtheabilityofImRePtoaccuratelyinfertheproportionofimmunecellsin

thesampled tissue.Wehypothesized that the fractionofB-andT-cells in thesamplewillbe

proportional with the fraction of receptor-derived reads in our RNA-Seq data. We used a

transcriptome-basedcomputationalmethod,SaVant16,whichusescell-specificgenesignatures

(independentofBCRorTCRtranscripts)toinfertherelativeabundanceofBorTcellswithineach

tissue sample. We found that B and T cell signatures inferred by SaVant showed positive

correlationwiththeamountofBCR(r=0.77,P<0.001)orTCR(r=0.86,P<0.001)transcripts,

respectively (Figure 2c,d). An exception to this correlation was for tissues that contain the

highestdensityofBorTcells:spleen,wholeblood,smallintestine(terminalileum),lung,and

EBV-transformedlymphocytes(LCLs).

chr2 chr7 chr14 chr22

IGL

TCRATCRD

IGH

TCRG

TCRB

IGKV N D N J C

CDR3CDR1 CDR2

MappedreadsUnmappedreads

mRNAencodingforTandBcellreceptorchains

VYFICASSEASSLGGSGGYTACCSYEQYFGPG

Vgene J gene

…SQTSVYFCASSE SYEQYFGPGTRLT…

CDR3

SQTSVYFICASSEASSLGGSGGY

Vgene…SQTSVYFCASSE

read

SGGYTACCSYEQYFGPG

J gene

SYEQYFGPGTRLT…

read1read2

TACCSYEQYFGPGYG

M

GM

LQ

! read2

Suffixtree

ImReP

a

b

c

d

readsmatchingonlyVgeneareencodedassuffixtree

Figure 1. Overview of ImReP. (a) Schematic representation of human adaptive immune repertoire. Adaptive

immune repertoire consists of four T-cell receptor loci (blue color, T cell receptor alpha locus (TCRA); T-

cell receptorbeta locus (TCRB);T-cell receptordelta locus (TCRD);andT-cell receptorgamma locus (TCRG))and

three immunoglobulin loci (red color, Immunoglobulin heavy locus (IGH); Immunoglobulin kappa locus (IGK);

Immunoglobulinlambdalocus(IGL).Alternativename–BCR,Bcellreceptor).B-andT-cellreceptorscontainmultiple

variable (V,greencolor),diversity (D,presentonly in IGH,TCRB,TCRG,violetcolor), joining(J,yellowcolor)and

constant(C,bluecolor)genesegments.V(D)Jgenesegmentsarerandomly jointedandnon-templatedbases(N,

darkredcolor)areinsertedattherecombinationjunctions.TheresultingsplicedT-orB-cellrepertoiretranscript

incorporatestheCsegmentandistranslatedintotheantigenreceptorproteins.RNA-Seqreadsarederivedfrom

the rearranged immunoglobulin IG and TCR loci. Reads entirely aligned to genes of B- and T-cell receptors are

inferredfrommappedreads(blackcolor).Readswithextensivesomatichypermutationsandreadsspanningthe

V(D)J recombinationare inferred fromtheunmappedreads (greycolor).Complementaritydetermining region3

(CDR3)isthemostvariableregionofthethreeCDRregionsandisusedtoidentifyT/B-cellreceptorclonotypes—a

group of clones with identical CDR3 amino acid sequences. (b) Receptor derived reads spanning V(D)J

recombinationsare identified fromunmappedreadsandassembled into theCDR3sequences.Wefirst scanthe

aminoacidsequencesofthereadanddeterminetheputativeCDR3boundariesdefinedbylastconservedcysteine

encodedby theVgeneand theconservedphenylalanine (forTCR)or tryptophan (forBCR)of Jgene.Given the

putativeCDR3boundaries,wechecktheprefixandsuffixofthereadtomatchthesuffixofVandprefixofJgenes,

respectively.(c-d)IncaseareadoverlapswithonlytheVorJgene,weperformthesecondstageofImRePtomatch

suchreadsbasedontheoverlapofCDR3sequenceusingsuffixtree.

Figure2.EvaluationofImReP.(a-b)EvaluationofImRePbasedonthenumberofassembledCDR3sequencesand

comparison to existing methods. (c) Concordance of targeted TCRB-Seq and non-specific RNA-Seq. (d-e)

CorrespondenceofImReP-derivedreadsfromB-cell(BCR)andT-cell(TCR)receptorstotherelativeabundanceofB-

andT-cellsinferredfromcell-specificgeneexpressionprofiles.(a)PrecisionandrecallratesforImReP(blue),MiXCR

(RNA-Seqmode) (blue), IMSEQ (green), and IgBlast (orange)on simulateddata for immunoglobulinheavy (IGH)

transcriptsarereportedforvariousreadslength(separateplots)andpertranscriptcoverages(1,2,4,8,16,32,64,128)

(x-axis).TRUSTandTraCeRdonotsupporttheanalysisofBCRsequencesandwereexcludedfromthecomparison

Final figures 2c,d

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r = 0.77P < 0.001

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CRA)

ImRePTCRBSeq MiXCR

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basedfortheIGHdata.(b)PrecisionandrecallratesforImReP(blue),MiXCR(RNA-Seqmode)(red),TRUST(default,

paired-endmode)(pink),TRUST(single-endmode)(violet),IMSEQ(green),andTraCeR(aqua)onsimulateddatafor

Tcellreceptoralpha(TCRA)transcripts arereportedforvariousreads length(separateplots)andpertranscript

coverages (1,2,4,8,16,32,64,128) (x-axis). (c) Concordance of targeted TCRB-Seq and non-specific RNA-Seq

performedonthreeTCGAsamples(onlyoneisshown)fromkidneyrenalclearcellcarcinoma(KIRC).Venndiagram

on TCGA-CZ-5463 sample presents number of matching CDR3s reported by immunoSEQ Analyzer

(http://www.adaptivebiotech.com/)andCDR3sequencesassembledfromnon-specificRNA-SeqdatabyImReP,

MiXCR(RNA-Seqmode),TRUST(default, paired-end mode). Results on other samples are presented in Figure

S4. (d)ScatterplotofthenumberofallBCRreadsper1millionRNA-Seqreads(y-axis)andB-cellsignaturescore

inferredbySaVant(x-axis).(e)ScatterplotofthenumberofallTCRreadsper1millionRNA-Seqreads(y-axis)andB-

cellsignaturescoreinferredbySaVant(x-axis).Pearsoncorrelationcoefficient(r)andP-valuearereported.

Characterizingtheadaptiveimmunerepertoireacross53GTExtissues

ImReP identified over 26million reads overlapping 3.8million distinct CDR3 sequences that

originatefromdiversehumantissues.ThemajorityofassembledCDR3sequencesderivedfrom

BCRs, with 1.7 million from the immunoglobulin heavy chain (IGH), 0.9 million from the

immunoglobulinkappachain(IGK),and1.0millionfromtheimmunoglobulinlambdachain(IGL).

AsmallerfractionofCDR3sequencesderivedfromTCRs,with0.2millionsequencesfromalpha

andbetaTCRs(TCRAandTCRB).ThevastmajorityofallassembledCDR3shadalowfrequency

inthedata.98%ofCDR3sequenceshadacountof lessthan10reads,andthemedianCDR3

sequencecountwas1.4.CDR3sequencesderivedfromIGKwerethemostabundantacrossall

tissues,accountingonaveragefor54%oftheentireB-cellpopulation(FigureS5).IntheT-cell

population,alphaandbetajointlyaccountedfor83%ofthepopulation.DeltaT-cellpopulation

wastherarest,accountingforlessthanonepercentoftheentireTcellpopulation(FigureS6).

We compared the length and amino acid composition of the assembled CDR3 sequences of

immunoglobulinandT-cellreceptorchains(Figure3a-g).Consistentwithpreviousstudies,we

observedthatimmunoglobulinlightchainshavenotablyshorterandlessvariableCDR3lengths

comparedtoheavychains17(Fig.3h).Thetissuetypeappearstohavenoeffectonthelength

distributionofCDR3sequencesofBCRs(FigureS7).DifferencesintheCDR3lengthdistributions

for TCRs cannot be estimated due to the small number of available TCRs. Sequencing

composition18ofCDR3regionsofbetaT-cellsassembledbyImRePrecapitulatesonedetectedby

TCRsequencing2fromthewholebloodwithtwodominantCASSandCSARmotifsinthebeginning

of the sequence (Fig. 3f). In line with other studies19, both light chains exhibited a reduced

amountofsequencingdiversity(Fig.3b-c).

Figure3.Lengthandaminoacidcompositionof theassembledCDR3sequencesof immunoglobulinandT-cell

receptorchains.Thesequencelogo(usingWebLogo)ofaminoacidcompositionrepresentationforCDR3sequences

withmean length.Theheightoftheaminoacidwithinthestack indicatestherelativefrequency.Distributionof

CDR3sequencelengthisestimatedusingskerneldensity.(a)Sequencelogoof15-amino-acidCDR3sequenceof

IGH.(b)Sequencelogoof11-amino-acidCDR3ofIGK.(c)Sequencelogoof12-amino-acidCDR3sequenceofIGL.(d)

Sequence logoof14-amino-acidCDR3sequenceofTCRA.(e)Sequence logoof14-amino-acidCDR3sequenceof

TCRB. (f) Sequence logo of 17-amino-acid CDR3 sequence of TCRD. (g) Sequence logo of 13-amino-acid CDR3

IGH

IGK

IGL

TCRD

TCRB

TCRA

a

h TCRG

b

c

d

e

f

g

Length(amino acids)

sequenceofTCRG.(h)DistributionofCDR3sequencelengthisestimatedusingskerneldensityseparatelyforeach

chainofT-andB-cellreceptors.

We observed per sample an average of 1331 distinct clonotypes for BCRs and 20 distinct

clonotypessequencesforTCRs.Wenormalizedthenumberofdistinctclonotypesbythetotal

numberofRNA-Seqreads,whichwecallnumberofclonotypesperonemillionreads(CPM).As

thenumberofdistinctclonotypesdoesnot increase linearlywiththesequencingdepth,CPM

metricshouldnotbeusedinstudiescomparingclonotypediversityacrossvariousphenotypes.

Instead,CPMisintendedtobeaninformativemeasureofclonaldiversityadjustedforsequencing

depth.

We used per sample alpha diversity (Shannon entropy) to incorporate the total number of

distinctclonotypesandtheirrelativefrequenciesintoasinglediversitymetric.Amongalltissues,

spleenhasthelargestB-cellpopulation,withamedianof1301BCR-derivedreadsperonemillion

RNA-Seqreads.ItalsohasthemostdiversepopulationofBcellswithmedianpersamplealpha

diversityof7.6correspondingto1025CPM(Figure4andTableS1).Wholebloodhasboththe

largestandmostdiverseT-cellpopulation(Figure4andTableS1).Organsthatpossessmucosal,

exocrine,andendocrinesites(n=24)harborarichclonotypepopulationwithamedianof87CPM

persample.Minorsalivaryglandshavethehighest immunediversity inthegroup(alpha=7.1)

andsurpassthediversityoftheterminalIleumcontainingPeyer’sPatches,whicharesecondary

lymphoidorgans(TableS1).

Tissuesnotrelatedtotheimmunesystem,includingadipose,muscle,andtheorgansfromthe

centralnervoussystem,containedamedianof6CPMpersample,whicharemostlikelydueto

thebloodcontentofthetissues20.ThehighestnumberofdistinctCDR3sequencesamongnon-

lymphoidorganswaspresent in theomentum,amembranousdouble layerofadiposetissue

containingfat-associatedlymphoidclusters.Asexpected21,Epsteinbarvirus(EBV)-transformed

lymphocytes(LCL)harboredalargehomogeneouspopulationofBcellclonotypes(TableS1and

FigureS7).

0 200 400 600 800 1000

CLONOTYPIC RICHNESSOFBCELLS, CPM

IGH IGK IGL

02468101214

SpleenSmallIntestine- TerminalIleumWholeBloodArtery- CoronaryArtery- AortaArtery- TibialMinorSalivaryGlandColon- TransverseStomachColon- SigmoidLungBreast- MammaryTissueEsophagus- MucosaThyroidKidney- CortexBladderFallopianTubeVaginaCervix- EndocervixLiverCervix- EctocervixProstateUterusOvaryPancreasPituitarySkin- NotSunExposed…Skin- SunExposed(Lowerleg)AdrenalGlandTestisCells- EBV-transformed…Cells- TransformedfibroblastsAdipose- Visceral(Omentum)Esophagus- Gastroesophageal…Adipose- SubcutaneousHeart- AtrialAppendageEsophagus- MuscularisHeart- LeftVentricleMuscle- SkeletalNerve- TibialBrain- Spinalcord(cervicalc-1)Brain- AmygdalaBrain- CortexBrain- HypothalamusBrain- Caudate(basalganglia)Brain- SubstantianigraBrain- HippocampusBrain- Putamen(basalganglia)Brain- AnteriorcingulatecortexBrain- Nucleusaccumbens(basal…Brain- CerebellumBrain- FrontalCortex(BA9)Brain- CerebellarHemisphere

TCRA TCRB

TCRD TCRG

a b

Secondarylymphoidorgans

Bloodassociatedsites

Mucosal,exocrineandendocrineorgans

Celllines

Adiposeandmuscleorgans

Organsofcentralnervoussystem

Tissuestypes

CLONOTYPIC RICHNESSOFTCELLS,CPM

Figure 4.Adaptive immune repertoires acrossmultiple human tissues. Adaptive immune repertoires of 8,555

samplesacross544individualsfrom53bodysitesobtainedfromGenotype-TissueExpressionstudy(GTExv6).We

groupthetissuesbytheirrelationshiptotheimmunesystem.Thefirstgroupincludesthelymphoidtissues(n=2,red

colors).Thesecondgroupincludesbloodassociatedsitesincludingwholebloodandbloodvessel(n=4,redcolor).

Thethirdgrouparetheorgansthatencompassesmucosal,exocrineandendocrineorgans(n=21,lavendercolor).

The fourth group are cell lines (n=3, grey color). The filth group are adipose or muscle tissues and the

gastroesophagealjunction(n=7,bluecolor).Thesixthgroupareorgansfromcentralnervoussystem(n=14,green

color).HistogramreportsclonotypicrichnessofTandBcells,calculatedasnumberofdistinctaminoacidsequences

ofCDR3peronemillionRNA-Seqreads(CPM).(a)MedianCPMsarepresentedindividuallyforTcellreceptoralpha

chain(TCRA),T-cellreceptorbetachain(TCRB),T-cellreceptordeltachain(TCRD),andT-cellreceptorgammachain

(TCRG).(b)MediannumberofdistinctaminoacidsequencesofCDR3arepresentedindividuallyforimmunoglobulin

heavychain(IGH),immunoglobulinkappachain(IGK),immunoglobulinlambdachain(IGL).

Individual-andtissue-specificT-andB-cellclonotypes

Aminoacidsequencesofclonotypesexhibitedextremeinter-individualdissimilarity,with88%of

clonotypesuniquetoasingleindividual(private)(Figure5a).Theremaining~400,000clonotypes

weresharedbyatleasttwoindividuals(public).Thenumberofindividualssharingclonotypes

variedacrossTandBcellreceptors,withimmunoglobulinlightchainshavingthehighestnumber

ofpublicclonotypes.Twenty-fivepercentofallIGKclonotypeswerepublic,andthenumberof

individuals sharing the IGK clonotype sequences can be as high as 471 (Figure 5b). T cell

clonotypeshadonaverage7%publicclonotypes,withthehighestnumberofpublicclonotypes

amongthedeltachainofgamma-deltaTCRs(12%).ThelimitedcapacityofRNA-Seqtocoverlow

abundant clonotypes may misclassify public clonotypes as private. Consistent with previous

studies10,22,weobservepublicclonotypestobesignificantlyshorterinlengththantheprivate

ones(p-value<2x10-16).Forexample, IGHchainpublicclonotypeshadanaveragelengthof13

aminoacids,andprivateclonotypeshadanaveragelengthof16.Wealsoexaminedwhetherthe

publicclonotypesweremoreoftensharedacrosstissueswithinanindividual.Only14%ofthe

~240,000 clonotypes shared across tissues were public. The majority of clonotypes were

individual-andtissue-specific(Figure5c).Thefulllistofpublicclonotypesisdistributedwiththe

‘AtlasofT-andB-cellrepertoires’thataccompaniesthismanuscript.

Figure5.PrivateandpublicTandBcellclonotypes.(a)Distributionoffrequenciesofprivate(n=1)andpublic(n>1)

clonotypesacross544 individuals.Wecollectclonotypes fromall tissuesof thesame individual intoa singleset

correspondingtothatindividual.(b)Themostpublicclonotypes(sharedacrossmaximumnumberof individuals)

and corresponding VJ recombination are presented for IGH, IGK, IGL, TCRA, TCRB, and TCRG. (c) Clonotypes

sequences are classified into public clonotypes (shared across individuals), private (individual specific), tissue-

specific,andclonotypessharedacrossmultipletissues.Thenumberofclonotypesfallingintoeachpairofcategories

isreportedacrossallTandBcellreceptorchains.

CQQYGSSPRTFIGKV3-20 IGKJ2471

CARGHYGMDVWIGHV3-53 IGHJ658

CQAWDSSTVVFIGLV3-1 IGLJ2443

CAGADRLQTGMRGAFTRAV8-7 TRAJ19

90

CVIKYFTRBV10-3 TRBJ2-367

CAAWDYYKKLFTRGV10 TRGJ1(2)44

a b

cTissuespecific Sharedacross

tissues

Individualspecific(private) 3.1x106 0.2x106

Sharedacrossindividuals(public)

0.4x106 0.03x106

-1 1 3 5 7 9 11 13 15 17Numbe

rofclono

type

sequ

ences

Numberofindividualssharing theclonotypesequence

IGH IGK IGL TCRA TCRB TCRD TCRG

106

105

104

103

102

10

1

BCRs

TCRs

FlowofTandBcellclonotypesacrosshumanGTExtissues

The large number of individuals available through the study allow us to establish a pairwise

relationshipbetweenthetissuesandtracktheflowofTandBclonotypesacrosshumantissues.

WobservedasignificantincreaseoftheCDR3sequencessharedacrosspairsoftissuesfromthe

same individuals. Further,weobserved thispattern consistently forall chainsofBandT cell

receptors(p-value<2x10-16)(Figure6aandTableS2).Weobserveadifferentamountofshared

CDR3sequencesacrossdifferent typesofBCRsandTCRswithan increase in immunoglobulin

light chains. Decreased number of TCR reads compared to the BCRs makes it unfeasible to

comparethenumberofsharedCDR3acrossTandBcells.Onaverage,weobserve21.0CDR3

sequencestobesharedacrossapairoftissuesfromthesameindividuals.Pairsoftissuesfrom

differentindividualsshareonaverage10.6CDR3sequences(Figure6aandTableS2).

ToestablishtheflowofB-andT-cellclonotypesacrossvarioustissues,wecomparedclonotype

populationsbetweenandwithinthesameindividuals.Welimitedthisanalysistopairsoftissues

forwhichwehadatleast10individuals(870pairsoftissuesoutof1378possiblepairs).Weused

betadiversity (Sørensen–Dicesimilarity index) tomeasurecompositional similaritiesbetween

thetissuesintermsofgainorlossofCDR3sequences(Figure6b-c).Forthemajorityofthe870

availabletissuepairs,weobservenoBCRorTCRsequencesincommon,whichcorrespondsto

betadiversityof0.0.

WeexaminedtheflowofIGHclonotypesacrosstissuesandpresenteditasanetwork(Figure

5b).Among870availabletissuepairs,wehaveidentified56tissuepairswithbetadiversityabove

.001.Spleenwasthemosthighlyconnectedtissue,with17connections,followedbylung,with

16 connections. Clonotypes represents one connected component, meaning that every two

nodesareconnecteddirectlyorviaothernodes.Clonotypepopulationsofspleenandlungare

themostsimilar (0.02betadiversity),otherpairs includeminorsalivaryglandandesophagus

mucosa,terminalileum(smallintestine)andtransversecolon.Weobserveabove200pairsof

tissueswithbetadiversityabove.001forimmunoglobulinlightchains(FigureS9-S10).Themost

similartissuepairsforIGKchainwerespleenandtransversecolon(0.15betadiversity).

WealsoexaminedtheflowofTCRBclonotypesacrosstissues.TCRBclonotypesequenceswere

sharedacross10pairsoftissueswithaveragebetadiversityof0.02.SimilartoBcells,thenetwork

of T cell clonotypes is a single connected component, with spleen being the most highly

connectedtissue(Figure6c).FortheTCRgammachain,weobservebetadiversityof0.0forall

tissuepairs.Atthesametime12%ofTCRgammaclonotypesarepublic,showingthehighestrate

amongallTCRgenes.AtthesequencingdepthprovidedbyRNA-Seq,weareunabletoobserve

TCRdeltaclonotypesharingacrossindividualsandtissues.

Figure6.FlowofTandBcellclonotypesacrossdiversehumantissues.Resultsarebasedonpairsoftissueswithat

least10individuals.(a)Thenumberofclonotypesequencessharedacrosspairsoftissuesfromthesameindividuals

(bluecolor)andfromdifferentindividuals(orangecolor)arepresented.Numberofclonotypessharedacrosstissues

fromthesameindividualsforTCRsis<.1.Numberofclonotypessharedacrosstissuesfromdifferentindividualsfor

TCRsis<.001.(b)-(c)Flowofclonotypesacrossdiversehumantissuesispresentedasanetwork.Eachnodeisatissue

withthesizeproportionaltoamediannumberofclonotypesofthetissue.Thecolorofthenodecorrespondstoa

typeofthetissuetype:lymphoidtissues(yellowcolors),bloodassociatedsites(redcolor),organsthatencompasses

mucosal,exocrineandendocrineorgans(lavendercolor).Compositionalsimilaritiesbetweenthetissuesintermsof

gain or loss of CDR3 sequences aremeasured across valid pairs of tissues using beta diversity (Sørensen–Dice

similarity index). Edges areweighted according to the beta diversity. (b) Flowof IGH clonotypes across diverse

ab

Averagenu

mbero

fclono

types

sharedacrossp

airsoftissues

c

IGH

TCRB

-15

-10

-5

0

5

10

15

IGH

IGK

IGL

TRCA

TRCB

TRCD

TRCG

Pairoftissueswithinthesame individual

Pairoftissues acrossdifferentindividuals

humantissuesispresentedasanetwork.Edgeswithbetadiversity>.001arepresented.(c)FlowofTCRBclonotypes

acrossdiversehumantissuesispresentedasanetwork.Edgeswithbetadiversity>.001arepresented.

ImRePidentifiestissuesampleswithlymphocyteinfiltration

Histologicalimagesoftissuecross-sectionsandpathologists’noteshavebeenusedtovalidate

the ImReP’s ability to detect the samples with a high lymphocyte content, which often

correspondstoadiseasestate.WeexaminedtheIGHclonotypepopulationsfromthyroidtissue

acrossindividuals.ThemediannumberofinferreddistinctCDR3sequencespersamplewas20,

though14.5%ofthesampleshadmorethan500distinctCDR3sequences. Weobservedthe

highestnumberofCDR3sequencesamongallthethyroidsamplesinanindividualwithlatestage

Hashimoto'sthyroiditis,anautoimmunediseasecharacterizedbylymphocyteinfiltrationandT-

cellmediatedcytotoxicity.Accordingtopathologists’notes,Hashimoto'sdiseasewaspresentin

11.2%ofthyroidsamples,withvaryingdegreesofseverity.First,weusedpathologists’notesto

annotate samples as healthy or bearing Hashimoto's disease, and then we compared the

adaptiverepertoirediversitybetweenthesegroups.Weobservedasignificant increaseinthe

numberofdistinct IGHclonotypes insampleswithHashimoto's thyroiditis (p-value=1.5x10-5)

(FigureS11).Thenumberofclonotypesvariedfrom113forfocalHashimoto'sthyroiditisto5621

forlatestageHashimoto'sthyroiditis(Figure7a).Inaddition,highclonotypediversityinkidney

samplesindicatedthepresenceofglomerulosclerosis.Inlungsamples,highclonotypediversity

correspondedtoinflammatorydiseasessuchassarcoidosisandbronchopneumonia.

Weobservednodifferenceinclonaldiversityinmalesandfemalesacrossthetissues,exceptin

breasttissues(p-value<3.2x10-12,BCRs). Increasedclonotypediversityofbreasttissue inmale

individualscorrespondedtogynecomastia,acommondisorderofnon-cancerousenlargementof

malebreasttissue(Figure7b).

Figure7.ImRePisabletoidentifysampleswithhighactivityoflymphocytes.Histologicalimagesoftissuecross-

sectionsandpathologists’noteshavebeenusedtovalidatetheImReP’sabilitytodetectthesampleswithahigh

activityoflymphocytes.(a)SampleswereorderedbyHashimoto'sthyroiditisseverity,asreportedbypathologists’

notes.Histologicalimagesareprovidedtoillustratethediseasestate.Averagenumberofclonotypesisreportedfor

eachdiseasegroup.(b)Boxplotreportingnumberofclonotypesinthebreasttissuesformalesandfemales.Outlier

amongthemalesamplesisillustratedwiththehistologicalimage.

113 4961315

2353

5621

FOCA

LHAS

HIMOTO

S

PATCHY

HA

SHIM

OTO

S

EARLYHA

SHIM

OTO

S

HASH

IMOTO

S

LATESTAGE

HA

SHIM

OTO

S

Num

berofIGH

clo

notypes

DiseaseseverityHistologica

limages

female

male

0

1000

2000

3000

Number of IGH clonotypes

sex_

s

Gynecomastia

b

a

Discussion

We have developed a novel computational approach (ImReP) for reconstruction of adaptive

immune repertoires using RNA-Seq data.We demonstrate the ability of ImReP to efficiently

extract TCR- and BCR- derived reads from the RNA-Seq data and accurately assemble

correspondingBCRandTCRclonotypes.TheproposedalgorithmcanaccuratelyassembleCDR3

sequencesofimmunereceptorsdespitethepresenceofsequencingerrorsandshortreadlength.

Simulations generated using various read lengths and coverage depth show that ImReP

consistentlyoutperformsexistingmethodsintermsofprecisionandrecallrates.

We have demonstrated the feasibility of applying RNA-Seq to study the adaptive immune

repertoire.AlthoughRNA-Seqlacksthesequencingdepthoftargetedsequencing(Rep-Seq),it

can compensate by examining a larger sample size. Using ImReP, we have created the first

systematicatlasofimmunologicalsequenceforB-andT-cellreceptorrepertoriesacrossdiverse

humantissues.Thisprovidesarichresourceforcomparativeanalysisofarangeoftissuetypes,

mostofwhichhavenotbeenstudiedbefore.TheatlasofT-andB-cellreceptors,availablewith

thepaper,isthelargestcollectionofCDR3sequencesandtissuetypes.Weanticipatethatthis

database will enhance future studies in areas such as immunology and contribute to the

developmentoftherapiesforhumandiseases.

Using RNA-Seq to study immune repertoires has some advantages, including the ability to

simultaneouslycapturebothTandBcellclonotypepopulationsduringasinglerun.Italsoallows

simultaneousdetectionofoveralltranscriptionalresponsesoftheadaptiveimmunesystem,by

comparingchangesinthenumberofBCRandTCRtranscriptstothemuchlargertranscriptome.

Given large number of large-scale RNA-Seq datasets becoming available,we look forward to

scalinguptheatlasofT-andB-cellreceptorsinordertoprovidevaluableinsightsintoimmune

responsesacrossvariousautoimmunediseases,allergies,andcancers.

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