Genomic organization of the zebrafish (Danio rerio)Tcell ...vetmed.tamu.edu/media/1777265/seelye imgt 2016 zebrafish tcr delta.pdfthe TCRα/δ locus of Danio rerio based on the most

ORIGINAL ARTICLE

Genomic organization of the zebrafish (Danio rerio) T cellreceptor alpha/delta locus and analysis of expressed products

Stacie L. Seelye1 & Patricia L. Chen1& Thaddeus C. Deiss1 & Michael F. Criscitiello1,2

Received: 11 October 2015 /Accepted: 18 January 2016# Springer-Verlag Berlin Heidelberg 2016

Abstract In testing the hypothesis that all jawed vertebrateclasses employ immunoglobulin heavy chain V (IgHV) genesegments in their T cell receptor (TCR)δ encoding loci, wefound that some basic characterization was required ofzebrafish TCRδ. We began by annotating and characterizingthe TCRα/δ locus of Danio rerio based on the most recentgenome assembly, GRCz10. We identified a total of 141 the-oretically functional V segments which we grouped into 41families based upon 70 % nucleotide identity. This numberrepresents the second greatest count of apparently functionalV genes thus far described in an antigen receptor locus withthe exception of cattle TCRα/δ. Cloning, relative quantitativePCR, and deep sequencing results corroborate that zebrafishdo express TCRδ, but these data suggest only at extremely

low levels and in limited diversity in the spleens of the adultfish. While we found no evidence for IgH-TCRδ rearrange-ments in this fish, by determining the locus organization wewere able to suggest how the evolution of the teleostα/δ locuscould have lost IgHVs that exist in sharks and frogs. We alsofound evidence of surprisingly low TCRδ expression and rep-ertoire diversity in this species.

Keywords Danio rerio . TCRα . TCR δ . γδ Tcells

Introduction

Zebrafish (Danio rerio) continues to increase in popularity avertebrate model species (Iwanami 2014). Zebrafish enteredthe forefront of animal research in the 1980s due to the abilityto perform large-scale genetic screens and production of de-velopmental mutants in the species with studies by GeorgeStreisinger (Chakrabarti et al. 1983; Walker and Streisinger1983). Over time, the use of the fish species was extended toother fields, such as pathology, toxicology, behavior, and evo-lution (Harper 2011). One significant area zebrafish has con-tributed is developmental and comparative immunogenetics(Iwanami 2014).

Understanding the organization of the genes that code forzebrafish lymphocyte antigen receptors is integral to our un-derstanding of the immune system of this useful animal mod-el. Tcell receptors (TCR), along with immunoglobulin, conferclonal specificity for activation of lymphocytes and are het-erodimers of two chains. The chains are typically divided intofour classifications, the α/β and γ/δ each forming pairs. Tcells bearing the γ/δ heterodimer have many subsets withunique properties and often exhibit features of innate immuneresponses. They are typically found in epithelial and gastroin-testinal tissues and are prevalent in early and fetal

This work was supported by the National Science Foundation through agrant to MFC (IOS 1257829).

Electronic supplementary material The online version of this article(doi:10.1007/s00251-016-0904-3) contains supplementary material,which is available to authorized users.

* Michael F. [email protected]

Stacie L. [email protected]

Patricia L. [email protected]

Thaddeus C. [email protected]

1 Comparative Immunogenetics Laboratory, Department of VeterinaryPathobiology, College of Veterinary Medicine and BiomedicalSciences, Texas A&M University, College Station, TX 77843, USA

2 Department of Microbial Pathogenesis and Immunology, School ofMedicine, Texas A&M University, Bryan, TX 77807, USA

ImmunogeneticsDOI 10.1007/s00251-016-0904-3

http://dx.doi.org/10.1007/s00251-016-0904-3

http://crossmark.crossref.org/dialog/?doi=10.1007/s00251-016-0904-3&domain=pdf

development in many species. Some γ/δ T cells migrate earlyin development to particular tissues such as the liver, skin,mucosa of the lungs, digestive, and reproductive organs andpersist as resident cells (Bonneville et al. 2010). γ/δ T cellsform a much larger proportion of the peripheral T cell pool inadult ruminants, rabbits, and chickens than in primates androdents (Hein and Mackay 1991; Holderness et al. 2013).Relatively little is known about the functional importanceand prevalence of γ/δ T cells in teleost fish. We do know thatthe physiological roles fulfilled by γ/δ Tcells in mammals arevaried. Some subsets of γ/δ T cells are unique in that theyrecognize conserved non-peptide antigens that are often up-regulated by stressed cells, the expression modalities and dis-tribution of which resemble those of pathogen associated mo-lecular patterns (PAMPs) or danger associated molecular pat-terns (DAMPs). This is in contrast to α/β T cells which arerestricted to recognizing and responding to peptide antigenpresented by self MHC molecules (Bonneville et al. 2010).During development of the thymocyte, TCR genes undergosomatic rearrangement of the genetic elements that encodecomponents of the receptor, called the variable (V), diversity(D), and joining (J) gene segments. There is considerable in-formation about V(D)J rearrangement and the facilitatingTCR locus organization in many mammalian species but thedata available for teleost and lower ectothermic vertebrates ismore sparse (Moulana et al. 2014). In most mammalian spe-cies the TCRδ locus is imbedded within the TCRα locus andhas the following arrangement: Vα/δ-Dδ-Jδ-Cδ-Jα-Cα, withsome V’s being used by both α and δ (Murphy 2012). Thegenomic arrangement for some teleost fish has been elucidat-ed, specifically for the Japanese pufferfish (Takifugu rubripes)(Wang et al. 2001b) and the green pufferfish (Tetraodonfluviatilis) (Fischer et al. 2002). These species show a uniqueorganization for teleost fish of Dδ-Jδ-Cδ-Jα-Cα-Vα/δ, withthe Vs in an inverted orientation with respect to the otherelements.

The genomic arrangement of the TCR αδ locus of someteleost fish has been elucidated, specifically for the Japanesepufferfish (T. rubripes) (Wang et al. 2001a) the greenpufferfish (T. fluviatilis) (Fischer et al. 2002), and theAtlantic salmon (Salmo salar) (Yazawa et al. 2008a). Thesespecies show a unique organization for teleost fish of Dδ-Jδ-Cδ-Jα-Cα-Vα/δ, with the Vs in an inverted orientation withrespect to the other elements. Additionally, the genes codingfor TCRδ and TCRα have been identified but the organizationof the genomic locus has yet to be elucidated in channel cat-fish (Ictalurus punctatus) (Moulana et al. 2014). There is alsolimited information about the TCRγ and TCRδ genes of themandarin fish (Siniperca chuatsi) (Tian et al. 2014), sea bass(Dicentrarchus labrax) (Buonocore et al. 2012), common carp(Cyprinus carpio) (Shang et al. 2008), and flounder(Paralichthys olivaceus) (Nam et al. 2003). To date, littlehas been published about the genomic organization of the

α/δ locus in zebrafish. Various papers have focused on indi-vidual aspects of these receptors and have found ample cDNAsequences coding for TCRα yet a very few TCRδ rearrange-ments (Haire et al. 2000) (Danilova et al. 2004) (Schorpp et al.2006).

Salmon have 128 potentially functional Vα/δ, human has57, mouse has 98, and chicken has 70. In zebrafish 148 Vαgenes have so far been found on BAC clones containing noapparent defect (Danilova et al. 2004). Previous work identi-fied two Vδ, two Jδ segments, three Dδ segments, as well asone Cδ segment (Schorpp et al. 2006). A cDNA library recov-ered four related TCRα clones, each with unique V, J, and Csequences and several Jα segments. The cDNA sequence forone Cδ and four Cα rearranged products has been identified(Haire et al. 2000).

An in t e r e s t i ng immunogene t i c phenomenonconcerning TCRδ has been discovered in nurse shark(Criscitiello et al. 2010), Xenopus (Parra et al. 2010b),chicken (Parra and Miller 2012), opossum (Parra et al.2008), platypus (Wang et al. 2011), and most recently inthe coelacanth (Amemiya et al. 2013). These vertebrateshave the ability to utilize immunoglobulin heavy chain(IgH) V gene sequences to create apparently functionalTCR δ (and perhaps α) chains. This process was origi-nally coined transrearrangement. Elucidation of the orga-nization of the TCRδ loci of some model species hasshown that many of these species possess V segmentslocated within the α/δ locus that show much higher iden-tity to immunoglobulin heavy chain V sequences than toTCRα/δ V sequences. In originally setting out to deter-mine if such IgHV segments are used in the teleostTCRα/δ loci, we annotated the zebrafish locus and foundevidence for low or at least unusual expression of canon-ical TCRδ.

Methods

C region search

A tBLASTn search was performed using the TCRδC se-quence from P. olivaceus (accession # BAC65463.1) againstversion GRC Z10 of the zebrafish genome (as well as otherbony fish genomes in our initial interrogation for IgHV genesegments). One match was found on chromosome 2. To verifythat this was TCRδC, this sequence was used to perform atBLASTn search against the nonredundant database. A phy-logenetic analysis of various mammalian, amphibian, teleost,and chondrichthyes species TCRδC sequences usingMUSCLE for multiple sequence alignment and the neighborjoining method to create a phylogenetic tree with bootstrapvalues from 1000 iterations was done using the MEGA 6.0software package (Tamura et al. 2013).

Immunogenetics

V region search

Using a previously annotated TCRδV sequence fromP. olivaceus (accession #AB076071.1) as bait sequence,a tBLASTn search was performed on the GRCz10 refer-ence assembly of zebrafish. Genomic sequences weredownloaded into the Geneious version 7 (Kearse et al.2012) software suite for annotation of the locus, multiplesequence alignments and phylogenetic tree analyses.Recombination signal sequences and intron splice signalswere identified manually for all sequences and were usedto determine the limits of the coding nucleotide se-quences of V, D, and J segments. All sequences weretrimmed to remove splice signals and recombination sig-nal sequences before a V gene multiple sequence align-ment was performed using Clustal W. A phylogenetictree was created using the neighbor joining method inMEGA 6.0 (Tamura et al. 2013). These V gene se-quences were analyzed using a percent identity matrixgenerated from the multiple sequence alignment. V seg-ments were placed in families based on the rule thatsequences that shared 70 % nucleotide identity with atleast one other sequence were placed in the same family.Families were then ordered based on their position with-in the locus. Groupings within families that showedhigher percent identity were placed in subgroups. Thiswas represented by the number after the first decimal inthe naming protocol. If the sequence did not belong in asubgroup, the second digit of 0 was used to denote nosubgroup.

Locus annotation

Scaffold version 10 of chromosome 2 was downloaded fromNCBI and imported into Geneious version 7. All 149V re-gions from the above BLAST search were annotated as wellas previously found D, J, and C regions for TCRα/δ. SomeTCRδ D, J, and C segments matched published sequences(Schorpp et al. 2006), previously described Jα sequences werefound using a custom annotation database created byIMGT/LIGM-DB (http://www.imgt.org/ligmdb/) andpreviously described Cα sequence was confirmed on thescaffolded assembly (Haire et al. 2000). Annotated sequenceswere then manually evaluated and the start and stop codonswere identified based on appropriate splice sites. The entirelocus was again visually inspected for the presence of addi-tional, potentially functional D and J segments. Additional Dsegments were analyzed for the presence of the heptamer,spacer, and nonamer sequence on both the 5′ and 3′ end ofsequences. Additional J segments were identified based on thepresence of the heptamer, spacer, nonamer sequence and thepresence of the FGxG motif as a hallmark of J regions as wellas the FGxP motif identified in zebrafish Jδ.

Search for evidence of TCRδ transcripts

A 5′ RACE library was created using the GeneRacer Kit(InvitrogenWalthamMA) from spleen RNA originally isolat-ed from 12 outbred zebrafish (kind gift from Matt Young).PCR was performed using a reverse primer to target theTCRδC region and forward primer to the GeneRacer 5′ oligoadapter. All primers used can be found listed in SupplementalTable 1. Primary PCR was performed using 1 μl 10 μMdNTP, 10 μl 5× Phusion buffer (New England Biolabs,Ipswich MA), 2.5 μl 10 μM GENERACER 5′ primer,2.5 μl 10μM reverse primerMFC527, 2 μl 50 ng/μl template,0.5 μl 2 U/μl High Fidelity Phusion DNA polymerase, andPCR quality water to total volume of 50 μl. Thermocycler(Bio-Rad C1000 thermal cycler, Bio-Rad Laboratories,Hercules CA) protocol was as follows: (1) initial denaturation95 °C for 15 min, (2) denaturation 95 °C 30 s, (3) annealingand elongation 72 °C for 30 s repeat steps 2–3 30 times, (4)final elongation 72 °C for 5 min. Secondary PCR was per-formed using 3 μl 25 mM MgCl2, 1 μl 10 mM dNTP, 1 μl10 μM GENERACER 5′ nested primer, 1 μl 10 μM reverseprimerMFC525, 3μl of template from primary PCR, 5 μl 10×Buffer, 0.25 μl 5 U/μl HotFire DNA Polymerase (SolisBioDyne, Tartu, Estonia), and PCR quality water to total vol-ume of 50 μl. Thermocycler protocol was as follows: (1) ini-tial denaturation 95 °C for 15 min, (2) denaturation 95 °C for30 s, (3) annealing 63 °C for 30 s 4) elongation 72 °C for1 min repeat steps 2–4 35 times (5) final elongation 72 °Cfor 5 min.

Amplicon DNA was extracted from the gel slice andligated into the PCR-II vector (Invitrogen). The plasmidwas used to transform chemically competent One ShotTop10 E. coli cells (Life Technologies, Waltham MA).Cultures were grown on ampicillin plates coated withX-gal. White and light blue colonies were selected andprepared for sequence analysis using the ZR PlasmidMiniPrep Kit (Zymo Research, Irvine, CA). Sequencingproducts from the plasmid were amplified using BigDye(Life Technologies) and samples were sequenced by theGene Technology Laboratory at Texas A&M. Geneiousversion 7 (Kearse et al. 2012) was used for sequenceanalysis.

Additional short, minimally degenerate primer PCR (Rastand Litman 1994) was performed targeting the conservedframework sequence encoding the WYRQ motif with thesame reverse primers from the 5′ RACE PCR. PCR was firstperformed according to the same protocol as mentioned abovefor the secondary PCR, with the primers used being 1 μl10 μM MFC535, MFC536, or MFC537 primers and 1 μl10 μM reverse primer MFC525. The amount of template usedwas 3 μl of 50 ng/μl 5′ RACE library cDNA. Thethermocycler protocol was also the same with the exceptionof using an annealing gradient of 30–54 °C for 30 s at step

Immunogenetics

http://www.imgt.org/ligmdb/

three and only 30 cycles were performed. No bands wereobtained from this attempt on 0.8 % agarose gel except forpositive control amplicons.

A second attempt was performed where the amount of25 mM MgCl2 was increased to 4 μl and only the primerMFC537 was used. The thermocycler protocol used was thesame as previous except with an annealing gradient 47–56 °Cfor 30 s and the number of cycles was increased to 35. Apositive control using 1 μl 10μM forward and reverse primersfor the housekeeping gene RpL13α under the same conditionswas included. The thermocycler protocol for the control wasthe same with the exception of using an annealing temperatureof 59 °C. Again, no bands were obtained on 0.8 % agarose gelexcept for positive control amplicons.

A third attempt was made where the amount of 25 mMMgCl2 was returned to 3 μl and the amount of the templatewas varied so that either 2, 4, 6, or 8 ng/μl of the 5′ RACElibrary template cDNA was used. The reverse primer waschanged to MFC 527. The remainder of the mixture was thesame. The thermocycler protocol used the same temperaturesused previously with the exception of the annealing tempera-ture of 52 °C for 30 s and 35 cycles were performed.Secondary PCR was performed using the reverse primerMFC525 and 1 μl of each of the primary PCR products. Theremainder of the components remained the same. The samethermocycler protocol was used with the exception of 30 cy-cles in this run. Again, no bands were obtained on 0.8 %agarose gel.

Quantitative real-time PCR

Quantitative PCR was performed with 50 ng of randomhexamer primed cDNA generated with SuperScript III fromRNA samples from pooled adult zebrafish spleen immunizedwith DNP-KLH via intraperitoneal injection (Weir et al.2015). We used the SYBR Green PCR Master Mix (Roche,Branford CT) following the manufacturer’s recommendation.Triplicate wells were assayed in a Roche LightCycler 480, for45 cycles annealing at 58 °C, followed by a melting curveanalysis. Primers for all four TCR constant region genes andthe ribosomal protein gene RpL13α are listed in SupplementalTable 1. The 2−ΔΔCt method using RpL13α as the calibrator(Livak and Schmittgen 2001) was used to calculate relativeTCR chain constant gene expression comparing unimmunizedcontrol fish to immunized fish. Summary statistics were per-formed in R with the summarySE function of Rmisc package(Hope 2013; R Core Team 2014). Statistical analyses of thevariance of mean, ANOVA, and Tukey HSD, were performedin R using the base stats package with a 95% confidence level(Chambers et al. 1992; R Core Team 2014). Visualization ofthe data performed in R with the ggplot2 package, statisticalsignificance indicated by p values of the Tukey HSD post-hoctest (Wickham 2009).

Pacbio sequencing

The same primers that successfully amplified the zebrafishTCRδ rearrangement above with the 5′ RACE approach werebarcoded for Pacbio SMRT deep amplicon analysis(Supplemental Table 1). cDNA was initially denatured at98 °C for 2 min then amplified with Phusion (NEB) highfidelity polymerase for 34 cycles consisting of two steps:98 °C for 10 s and 72 °C for 40 s, ending with a final elonga-tion at 72 °C for 5 min. Bands were excised after visualizationin a 1 % agarose gel, and extracted using Qiaquick gel extrac-tion columns (Qiagen). Samples were pooled and sent to DukeUniversity Genome Sequencing Center for Pacbio small insertlibrary preparation (1–3 kb) and SMRT sequencing (P6-C4Chemistry). Initial quality control, read filtering, andCircular Consensus Sequence (CCS) analysis were performedat the Duke University Genome Center. CCSs containing theproper barcoded primers were then annotated within theGeneious R7 Software Suite (Biomatters).

Results

In order to analyze canonical TCRδ use in zebrafish andsearch for Ig/TCR transrearrangements in the teleosts, theTCR α/δ locus of D. rerio was manually annotated usingthe latest genome assembly and taking into account thescant expression data in the literature, sequence databases,and PCR cloning in our laboratory. This resulted in thefirst complete map of the locus and the description ofmany previously undescribed genetic elements (Fig. 1).The general organization of the locus follows that of otherteleosts studied: Dδ-Jδ-Cδ-Jα-Cα-Vα/δ. The gene names,functionality, genomic sequences, and deduced amino ac-id sequences of all V, D, J, and C segments for TCR αand δ are found in Supplemental Data 1.

Constant region

A putative zebrafish TCR delta C gene at 36,107,203-36,108,481 of chromosome 2 showed 100 % identity to the one iden-tified on clone DKEY-161 L11 (accession #BX681417) ofzebrafish linkage group 2 (Schorpp et al. 2006), a 56 % iden-tity match with the TCR delta C of carp and 42 % with that ofsalmon. Based on this homology paired with weaker identityto other TCR chain C genes and expression in transcripts withD and J segments 5′ of this C gene, we annotate this asTCRDC. A neighbor joining phylogenetic tree with bootstrapvalues supporting that this gene is indeed TCRδ(Supplemental Figure 1). The multiple sequence alignmentused to create this tree is shown in Supplemental Figure 2.No additional potential Cδ sequence locations were found inthe zebrafish genome.

Immunogenetics

36,492,580

36,512,209

36,610,354

36,198,145

36,178,516

36,158,887

36,139,258

traj106 traj107 traj108 traj109 traj110 traj111 trac tractrac

36,394,435

36,335,548

traj75

36,315,919

36,296,290

tradv23.2.8 tradv33.0.9 tradv33.0.10tradv36.1.2

36,276,661


36,590,725

36,571,096

36,531,838

36,551,467

36,472,951

tradv36.1.5 tradv23.3.3tradv33.0.13 tradv34.0.5

36,453,322

traj76

36,433,693

36,414,064

36,257,032

36,237,403

36,217,774

tradv3.0.1 tradv4.0.1 tradv4.0.2 tradv2.0.2tradv3.0.2


tradv6.0.1 tradv7.0 tradv6.0.3 tradv2.0.4tradv6.0.2 tradv6.0.4

tradv5.0.2 tradv8.0 tradv9.0.2 tradv10.0.1tradv9.0.1

tradv11.0.1 tradv12.0.1 tradv11.0.2 tradv10.0.3tradv10.0.2 tradv2.0.2

tradv10.0.4 tradv11.0.3 tradv13.0 tradv10.0.5

tradv14.0 tradv15.0 tradv17.0 tradv18.0tradv16.0

tradv30.1.5 tradv23.3.4 tradv36.2.4

traj77

tradv30.1.7 tradv23.3.6 tradv36.2.7 tradv34.0.6

tradv36.2.5 tradv36.2.6tradv23.3.5

36,374,806

36,355,177

tradv31.0 tradv23.1.4 tradv32.0.1 tradv23.1.6tradv23.1.5



tradv23.1.10 tradv30.0.5tradv34.0.2 tradv33.0.8tradv30.1.4

traj78

36,050,000

tradv23.2.2

36,119,629

36,100,000

traj11traj9traj6traj1 traj2 traj3 traj4 traj5 traj7 traj8

36,655,000

tradv23.2.3 tradv30.1.5

tradv35.0 tradv33.0.3 tradv23.2.1

traj30traj28 traj29traj27traj25 traj26traj23 traj24traj22traj20 traj21traj19traj18traj17traj15 traj16traj14traj13traj12 traj31

trdctrdctrdctrdj2trdj1trdd6trdd2trdd1 trdd5trdd4 trdd3

traj10



Nico�namide nucleo�de adenylyltransferase 2

tradv23.0.4 tradv25.0.4 tradv27.0

traj79

//

P21protein (Cdc42/Rac)-ac�vated kinase 2a

tradv40.0 tradv41.0

tradv28.0 tradv29.0 tradv30.0.1

tradv33.0.4

tradv36.1.1 tradv33.0.5 tradv37.0 tradv38.0.1

tradv39.0 tradv30.0.2tradv38.0.2


traj80




tradv32.0.4 tradv36.2.10tradv36.2.9

tradv3.0.3 tradv4.0.3 tradv2.0.3 tradv5.0.1tradv3.0.4

tradv1.0 tradv2.0.1

traj81


tradv30.1.2 tradv30.1.3tradv30.0.3 tradv30.0.4tradv23.3.2

tradv19.0 tradv20.0 tradv23.0.1 tradv10.0.3tradv21.0 tradv23.1.1tradv22.0


tradv25.0.1

tradv26.0 tradv24.0.3 tradv25.0.2


traj82

tradv30.1.6

traj83 traj84 traj85 traj86



traj105traj103 traj104traj102traj100 traj101traj98 traj99traj97traj95 traj96traj94traj93traj92traj90 traj91traj89traj88traj87

traj72 traj73 traj74

Inhibitor of growth isoform X1

Fig. 1 Annotation of TCR α/δ locus. This annotation is based on theversion 10 assembly for Chromosome 2, accession number NC_007113,released on September 24, 2014. Yellow arrows in δ D region representexpressed sequences and orange represent potential segments. Light bluearrows in α J region represent expressed sequences and dark blue arrowsrepresent potential segments. Red ovals represent V sequences expressed

with TCR δ constant region. Blue ovals represent V sequences expressedwith TCRα constant region. Arrows represent transcriptional orientation.The numbers represent the nucleotide designation at the beginning ofeach row. The last annotation represents the last nucleotide for theP21protein (Cdc42/Rac)-activated kinase 2a

Immunogenetics

V regions

Initial tBLASTn searches with flounder Vδ sequence revealed159 sequence matches on chromosome 2. Of these 147 se-quences were located downstream of the putative Cα regionranging from nucleotides 36,200,000 to 36,600,000. Twelvewere discounted as being too many megabases away to beused in this locus. Five sequences (tradv23.2.8, tradv30.1.5,tradv36.2.8, tradv36.2.9, and tradv36.2.11) were incompleteand after visually inspecting the genomic sequence, it wasdetermined that they should be classified as pseudogenes asthey did not contain appropriate splice sites. Two additionalsequences were removed from consideration due to one beingan overlapping duplicate and the second sequence showingonly minimal sequence homology to the other V sequences.Three contained stop codons (tradv12.0.2, tradv12.0.1,tradv23.2.6). Four additional V sequences (not of the original159 identified by BLAST) were found using a custom anno-tation database created by IMGT/LIGM-DB (http://www.imgt.org/ligmdb/). This gives a total of 141 theoreticallyfunctional Vα/δ segments and 8 pseudogenes in this locus.All 141 of these sequences have the canonical sequence struc-ture; the conserved cysteine residues necessary forintradomain disulfide bonds, conserved WYXQ motif in theFR2 region, the YYCA motif in FR3, as well as the RSSlocated at the 3′ end of each coding segment.

Next, an analysis of these V region genes was performed.Based on a percent identity matrix (Supplemental Table 1)created from a Clustal W (Tamura et al. 2013) multiple se-quence alignment (Supplemental Figure 3), these 141 V se-quences were placed into 41 different families. Of these, 23represented single gene families. Three of these families(tradv23, tradv30, and tradv36) were further annotated intosubfamilies. The sequences in subfamily tradv23.1 andtradv23.3 all have at least 70 % nucleotide identity betweeneach other sequence of these respective subfamilies (Pascualand Capra 1991). There are two members of subfamiliestradv23.2 and tradv23.3 that share greater than 70 % identityand six sequences in subfamilies tradv23.1 and tradv23.2whose identity is above that threshold. There are no sequencesbetween tradv23.1 and tradv23.3 that share 70 % identity butthey are linked by their similarity with subfamily tradv23.2,hence they were all placed in the same family. Sequences fromsubfamily 30.1 share 70 % nucleotide sequence identity witheach additional sequence in the subfamily. Sequences withdesignation 30.0 have 70 % identity to only some of the othersequences in the subfamilies 30.0 and 30.1, but not each se-quence. Within subfamilies tradv36.1 and tradv36.2, eachgene again has 70 % nucleotide sequence identity to eachadditional sequence within these respective subfamilies, butbetween these two subfamilies, there are four sequences thatshare 70 % or greater identity, justifying their placement in thesame family.

A neighbor joining tree was created from a Clustal W(Tamura et al. 2013) multiple alignment using the Mega 6.0software (Tamura et al. 2013). This tree (Fig. 2) confirmed ourplacing of sequences into families and subfamilies from thepercent identity matrix. While the tree is based on the nucle-otide sequence alignment in Supplemental Figure 3, the aminoacid sequence for these V regions is found in SupplementalFigure 4.

D and J segments

There were three new Dδ and no new potentially functional Jδsegments identified to add to the three Dδ and two Jδ seg-ments already identified (Schorpp et al. 2006). There were anadditional 94 Jα segments located, bringing the total to 111when including the 17 Jα sequences found using a customannotation database created by IMGT/LIGM-DB (http://www.imgt.org/ligmdb/) which references previouslyunpublished work by Hohman et al. and submitted to NCBIGenbank in 2001 (Genbank accession numbers AF424544,AF424545, AF424546, AF424547, AF424548, AF425590,and AAL29405.1) and Hammond from the Wellcome TrustSanger Institute (Genbank accession number AL591399). Allof these segments had the canonically accepted sequencestructure where the D sequences can be read in all threeframes and RSS at both the 5′ and 3′ end. The Jα sequencescontain the conserved FGxG motif and a 5′ RSS were alsofound for each sequence. These sequences and their respectiveRSSs consisting of a conserved heptamer, 12/23 spacer andnonamer are shown in Supplemental Figure 5.

PCR cloning of TCRδ cDNA products

Multiple PCR strategies from cDNA of multiple fish onlyyielded one TCRδ rearrangement. An alignment of the oneselect clone and two additional sequences previously pub-lished is shown in Fig. 3. An alignment of all 8 of the originalclones obtained by plasmid transformation and Sanger se-quencing is shown in Supplemental Figure 6.

No successful PCR amplification was obtained from min-imally degenerate primers to avoid any inefficiencies inRACERNA adaptor ligation targeting the conserved V frame-work sequence. PacBio sequencing revealed an additional 440clones. All clones had identical V sequences tradv23.2.2 aswell as identical CDR3 regions. An alignment of all 440clones is shown in Supplemental Figure 7.

Quantitative real-time PCR of four TCR chain genetranscripts

The paucity of cloned functional TCRδ transcripts by tradi-tional PCR prompted us to use quantitative real-time PCR toanalyze the levels of change upon immunostimulation of the

Immunogenetics





four TCR chain transcripts (Fig. 4). Not only were the relativeincreases in both transcripts required for the γδ TCR hetero-dimer very low compared to TCRα, so was that of theβ chainof the αβ receptor. At least in the spleen of immunizedzebrafish the levels of TCRα upregulation at the mRNA levelappears much higher than the other three TCR chains and mayexplain the lack of TCRδ expressed gene rearrangements wefound, as both TCRγ and TCRδ transcripts appear to be

limiting. Cloning of the quantified amplicons for sequenceconfirmation is shown in Supplemental Figure 8.

Discussion

In testing the hypothesis that all jawed vertebrate classes haveintegrated immunoglobulin heavy chain V gene segments in

tradv

3.0.

3tra

dv3.

0.2

56

tradv

3.0.

1

99

tradv

3.0.

4

99

tradv

4.0.

1tra

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0.3

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241

92

72

tradv

7.0

42

tradv

6.0.

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0.1

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37

55

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14

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24

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98

17

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Fig. 2 Phylogenetic analysis of all genomic Vα/δ sequences fromD. rerio. The neighbor joining tree was drawn using MEGA 6.0 and1000 bootstrap replications. Colored asterisks represent differentphylogentic families (some colors were re-used due to limitations in

number of colors.) Triangles represent those sequences known to beexpressed with Cδ. Circles represent those Vs known to be used withCα. Sequences, expression data, and nucleotide locations for start andstop codons are found in Supplemental Data 1

Immunogenetics

their TCRδ encoding loci and TCRδ repertoires in a dominantteleost model species, we found that much basic characteriza-tion was required of zebrafish TCRδ and the genomic andexpressed mRNA levels of this gene. Despite the growingpopularity ofD. rerio as an animal model, there was a surpris-ing scarcity of information detailing the genetic organizationand expression data surrounding their use of γδ T cells.Seminal early work described TCRα products of the zebrafishα/δ locus (Haire et al. 2000), and described 8 Vα families thatare highly expressed (Danilova et al. 2004). This later publi-cation refers to unpublished work by T. Ota and the SangerCenter identifying at least 148 Vα sequences that have beengrouped into 87 families. These genomic annotations weresubmitted to NCBI Genbank with ascension numbersclone101L20 (Genbank Accession Number AL591481.5),clone 71H18 (AL596128.9), clone 18F12 (AL592550.11),

c lone 172D23 (AL591399 .3 ) , and c lone 40G1(AL591674.3). However, no further information was provid-ed about the criteria used for these family groupings or theirposition on the genome assembly. A third formative paperprovided the first look into the genetics of TCRδ (Schorppet al. 2006). This paper provided the genomic coding se-quence for three Dδ, two Jδ, and one Cδ gene segments froma BAC library (GenBank accession number BX681417.10).Interestingly the Jδ genes use an FGxP motif instead of themore common FGxG amino acid motif, where there is a pro-line substituted in place of a second glycine in the di-glycinebulge. These works only reported one complete TCRδ V re-arrangement from zebrafish (Schorpp et al. 2006), and onemore was found in the NCBI database (Fig. 4). Here, wecompleted the α/δ locus annotation, finding no evidence forIgH V segments. We attempted to analyze the expressed

< Leader >< V sequence 23.2.2 M E K Q L M L I L I L T P G V M T A D Q I S P N K E A L T V K EC1 ATGGAGAAA------CAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCCAAATAAAGAAGCTCTTACTGTAAAGGAAG< Leader >< V sequence 23.2.1

M E K Q L M L I L I L T P G V M T A D Q I R P N K E A F T V K EB35 ATGGAGAAA------CAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGGCCAAATAAAGAAGCTTTTACTGTAAAGGAAG< Leader >< V sequence 23.3.3 M E R G F L L V V V V I M A T G L V F G D N I E P E E K D V M T K EEST ATGGAAAGAGGCTTTCTACTCGTTGTTGTTGTTATTATGGCGACAGGTTTGGTATTTGGGGACAATATTGAGCCAGAGGAGAAAGATGTTATGACAAAAGAAA

E E T V T F S C S Y D T S S S Y V R L Y W Y R Q Y L N G E P Q Y L L FC1 AGGAGACAGTGACCTTCAGTTGCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTATTTATTATT E E T V T F S C S Y E T S S S Y V W L Y W Y R Q H L N G E P Q Y L I FB35 AGGAGACTGTGACCTTCAGCTGCTCATATGAAACAAGCAGCAGTTATGTTTGGCTTTACTGGTACAGACAGCATCTTAACGGAGAACCTCAGTATTTAATCTT R E A V K L A C S Y S T T N N R V R L Y W Y R Q N P N A E L L L L T YEST GAGAAGCTGTCAAGCTGGCCTGCTCATACAGTACAACCAACAATAGAGTTCGGCTTTATTGGTACAGACAGAATCCAAATGCAGAACTTTTGCTTTTAACATA

K A A R S S S G G G R P D N P R F K S T T S D S S T E L T I S G V TC1 CAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCACTATTAGCGGTGTAACT K P A K S A S V S G D P V D R R F Q S S T S D S S T E L T I S G A TB35 TAAACCTGCAAAATCAGCTTCTGTAAGTGGAGATCCAGTTGATCGTAGGTTTCAGTCGAGCACATCAGACTCATCCACTGAACTCACTATTAGCGGTGCAACT K G A R S L S A K H S S N D R F Q S T T S D S S T E L T I T D V R EST CAAAGGTGCTCGATCTCTGAGTGC---TAAACACTCCTCTAATGATCGGTTTCAATCCACAACATCAGACTCATCCACTGAACTCACTATTACTGATGTGCGT

V sequence > < N/P >< D1 > < D4 > < D6 > < N/P > TACTTGGGAC GATTGGGGTAC TCTGGACTAC L S D S A L Y Y C A L R V G E Y D Y C1 CTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAG A GTAC GACTAC L S D S A L Y Y C A L R V G T W D D W G T G H AB35 CTGTCAGATTCAGCTCTCTATTATTGTGCTCTTAGAGTTGG TACTTGGGAC GATTGGGGTA CTGGAC ATGCC L S D S A L Y Y C A L R V G V G V R VEST CTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAG TTGGGGTAC GGGTT

< J2 A T D P L T F G K P I T L T V I PC1 ---GCTACTGATCCTTTAACATTCGGCAAACCGATCACCCTCACGGTAATACCA-Cδ

< J1 D P L T F G A P I R L T V N P B35 ---------GACCCACTAACTTTCGGAGCTCCCATCCGTCTCACGGTCAATCCA-Cδ

< J2 S A T D P L T F G K P I T L T V I EST EST TCAGCTACTGATCCTTTAACATTCGGCAAACCGATCACCCTCACGGTAATACCA-Cδ

Fig. 3 Alignment of all three known expressed zebrafish Vδ sequences.Differences in Vand J sequences are highlighted in yellow. Differences inD and N/P nucleotides were not highlighted. Conserved hallmarksequences were highlighted in gray. Blue, green, and magentahighlighting marks the nucleotides and amino acids encoded by D1, D4

and D6, respectively. C1 was selected sequence from clones obtainedfrom zebrafish spleen in this work. Sequences were from databasesearch in NCBI yielding the EST (#DT064263.1) and B35 was fromthe literature (Schorpp et al. 2006)

Immunogenetics

TCRδ repertoire in the fish to rule out transrearrangements todistant IgH V segments, however we found isolation of evencanonical (TCRδV-TCRδD-TCRδJ-TCRδC) TCRδ tran-scripts a challenge, only finding one (and no IgHV-TCRδD-TCRδJ-TCRδC products).

Locus organization

The unusual locus organization with α/δV genes in aninverted transcriptional orientation 3′ of TCRαC we foundin D. rerio has also been observed in Tetraodon nigroviridis(Fischer et al. 2002), T. rubripes (Wang et al. 2001b), andS. salar (Yazawa et al. 2008b). There is no locus organizationdata available regarding other teleost fish, only expressiondata based on cDNA analysis. This may be important evolu-tionarily as analysis of the skate TCR α and δ loci showsevidence of a larger linkage distance than seen in mammals(Rast et al. 1997). The difference seen in transcriptional ori-entation for the various segments, suggests that teleosts usemore inversional recombination to generate their T cell α andδ functional Vencoding exons than the deletional recombina-tion that is commonly seen in mammals (Fig. 5a). When se-quences are in opposite orientation, recombination results ininversion of the gene segments instead of deletion (Agard andLewis 2000). Importantly, this organization does not delete theδ locus at first functional α rearrangement, as is the case in

most vertebrates. Thus other mechanisms (possibly greaterErk influence) must control ultimate γδ versus αβ lineagecommitment in teleosts.

Further exploration of the locus organization of additionalteleost species as well as other cartilaginous and bony fish iswarranted as this organization may give additional insight inthe evolution of the αδ T cell receptor locus and T lineages.The use of IgHV on TCRδ seemed to be an immunogeneticdevice evolved in shark and maintained in many vertebrategroups (Criscitiello 2014), yet apparently teleosts discarded itas did several endotherm lineages. It seems possible that anincomplete recombination of the Varray in an ancestral teleostto the other side of the D-J-C exons could have produced thedownstream V’s absent the IgHVs seen in many other verte-brates (Fig. 5b). Duplications within this hypothesized ances-tral shark organization could explain the distinct organizationin the amphibian Xenopus (Parra et al. 2010a). In consideringthe use of IgHV segments in the TCRδ repertoire of sharksand frogs yet so far not fish, is it possible that this hypothe-sized inversional locus reorganization in an ancestral teleost isresponsible for the loss of the IgHV-TCRδ chimeric receptorsin fish? More comparative loci analysis is needed and in themeantimewe suggest such a model (Fig. 5b). Starting with thelocus organization as we understand it in cartilaginous fish,recombination moving δD-δJ-δC-αJ-αC to a location to theother side of the αδVarray could have disrupted the syntenyof the IgHV segments with the Vs of the locus yielding theorganization and lack of IgHV seen in zebrafish. From theshark organization tandem duplication of many elements andmovement of duplicated blocks could yield the much morecomplex locus seen in Xenopus. Deletion of the IgHV/α/δV-δJ-δC-αJ-αC center of the locus organization seen in theanuran amphibian would result in the genomic organization ofthe TCRαδ locus seen in most mammals.

There also appears to be only a single TCRδ locus in thezebrafish. This is based on our BLAST search results thatrevealed only one matching genomic sequence in theD. rerio genome. The 5′, δD end of the locus is flanked bythe nicotinamide nucleotide adenylyltransferase 2 gene inzebrafish as it is in salmon. This is similar to what is thus farfound in cartilaginous fish, and higher vertebrates. There are afew exceptions to this rule however. For instance, theP. olivaceus was found to possess a second Cδ sequence thatexisted within the Cγ gene locus (Nam et al. 2003).Additionally, the occurrence of a second TCR δ locus is seenin the Galliforms, such as chickens and turkeys (Parra et al.2012b). In addition to the conventional TCR α/δ locus, theyhave a second TCRδ lineage that is unusual in that the V genesare more related to IgHV genes than to TCRV genes. There isevidence that both loci can be active as there is evidence ofexpression of traditional TCRδ receptors as well as those thatutilize the IgHV gene with the TCRδ constant region, similarto the transrearrangement phenomenon seen in sharks and the

Fig. 4 Splenic TCR chain relative expression preference. qPCR for eachTCR chain from zebrafish splenic cDNA. Center lines represent meanrelative change in expression, boxes represent a 95 % confidence intervalcentered on the mean, and whiskers represent standard deviation from themean. Statistically significant variances in chain expression weredetermined through ANOVA and post-hoc Tukey HSD

Immunogenetics

chimeric receptors in frogs. This second locus is not foundelsewhere, including in other avian lineages such as thePasseriformes (Parra and Miller 2012). There is strong evi-dence (Parra et al. 2012a) that the IgHV genes used inTCRδ loci and the plasticity in TCRδV use facilitated theevolution in monotremes and marsupial mammals of an addi-tional fifth TCR chain (TCRμ) that is distantly related toTCRδ (Parra et al. 2007; Wang et al. 2011).

D, J, and C segment analysis

While analyzing and identifying the J segments for TCRδ, itwas noticed that these J sequences did not contain the hall-mark FGxG sequence; instead, the second glycine is replacedwith a proline. This same substitution was also found in thecatfish (Moulana et al. 2014). The Atlantic salmon containsone functional Jδ that utilizes a FGKA sequence (Yazawaet al. 2008b) while Tetraodon utilizes the canonical FGxGmotif (Fischer et al. 2002).

The complete TCRδ C protein sequence found in zebrafishshows high sequence homology to the TCRδ C sequences ofother teleost fish, particularly with that of the common carp(C. carpio) and S. salar, with 56 and 42 % amino acid identityrespectively. TCRδ C sequences have also been found forother teleost species including the channel catfish, Japaneseflounder, puffer, and fugu. Based on the phylogenetic tree inSupplemental Figure 1, all of these sequences do show highhomology with each other and usually to TCRδ C of otherclasses than to any of the other TCR chains (TCRα of closelyrelated fish being the exception). In addition to its high ho-mology to other TCRδ C, the D.rerio TCRδ C does containthe highly conserved cysteine residues.

V region analysis

Zebrafish appear to have the second largest number of poten-tially functionalα/δV genes of any species previously studied(Fig. 6). The species with the greatest number of V sequencesis the cow (Connelley et al. 2014), which curiously has a

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Fig. 5 TCRα/δ locus organization has rearrangement consequences andevolutionary insights. a The zebrafish locus can rearrange with an initialV-D or V-J inversion for TCRδ or TCRα respectfully, yet both of theseprocesses leave open the possibility of subsequent rearrangements formaking the other TCR chain, and even both. bWith what we now knowabout teleost, frog, endotherms, and preliminary data in elephantshark (Venkatesh et al. 2014) and nurse shark (data not shown), it ispossible to hypothesize the inversions, duplications and deletions thatcould have shaped the TCR α/δ locus organization in different

vertebrate classes. Pointed ends of the pentagons representing V, J, andC gene segments denote transcriptional orientation. Red circles are signaljoints left in genome by inversional V(D)J recombination. Gray arrowsand brackets denote inversions and block movements, bowties markdeletions. Placental mammals refers to Infraclass Placentalia,monotremes such as the platypus do have IgHV in their TCR α/δ locus(Parra et al. 2012a) and use a choriovitelline placenta that providesnutrients primarily from the yolk sac

Immunogenetics

greatly restricted IgHV repertoire but diversifies someultralong CDR3H into an additional diverse microdomain(Wang et al. 2013). The species with the next highest numberof TCR α/δ V segments after zebrafish is S. salar whichpossess 128 potentially functional out of a total of 292 Vgenes, the remainder being pseudogenes (Yazawa et al.2008b). This salmon study goes on to compare the numberof α/δ V genes in S. salar to the number in chicken (Gallusgallus) (70), human (Homo sapiens) (57), and mice (Musmusculus) (98). An exhaustive search for the number of Vsegments has not been conducted in other teleost species tomake a valid comparison of these numbers. T. nigroviridis hasonly 13 Vα/δ segments (Fischer et al. 2002), but this specieshas a condensed genome, so this is not surprising. A total of21 distinct V sequences have been found by cDNA sequenc-ing in I. punctatus (Moulana et al. 2014) but this has not beentraced back to the number of segments at the locus so this may

not be an adequate representation of the number of V seg-ments at the genomic level.

In comparison to S. salar, D. rerio appears to possess asubstantially lower number of pseudogenes (9 versus 164).However, when comparing the number of pseudogenes inpufferfish (0), mice (9), and humans (11) (Yazawa et al.2008b), it does appear that S. salar are the outlier with anunusually high number of pseudogenes.

The 149α/δV sequences that were located could be placedin 41 families based on 70 % nucleotide identity. Previousunpublished work by T. Ota placed 148 V genes into 87 fam-ilies, but only 58 of these are in Genbank and no furtherinformation was available on their annotation or assignmentto families (Danilova et al. 2004). Based on the percent iden-tity matrix, there were some sequences that showed 70 %identity to some other sequences in a group but not to all.For the sake of clarity, we defined the family to include all

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Fig. 6 Functional α/δV genes indifferent vertebrates. Graphcompares the number of V α/δsegments found in the germlineDNA to the number of those thatare thought to be functional and tothe number of V segments thathave been found to be expressedwith α, δ, or both. Cattleinformation can be found in http://www.imgt.org/IMGTrepertoire/Proteins/index.php#C and(Connelley et al. (2014), Atlanticsalmon in (Yazawa et al. (2008b),zebrafish information can be infound in Supplemental Data 1 ofthis manuscript, mouse andhuman information can be foundin http://www.imgt.org/IMGTrepertoire/Proteins/index.php#C, and Tetraodon in (Fischeret al. 2002)

Immunogenetics

http://www.imgt.org/IMGTrepertoire/Proteins/index.php%23C






sequences that share 70 % nucleotide identity with at least oneother sequence in that family. We chose 70 % as our cutoffbased on the guidelines put forth by IMGT (http://www.imgt.org). Numerous papers have utilized various methods forclassifying V sequences into families. Inconsistency innaming and classifying V sequences may potentiallycompound our ability to compare the number of families andcharacteristics of these families across various species ofteleost fish. Yazawa et al. utilized 70 % nucleotide identityto classify the 292 V segments of S. salar into 62 families.But this is far from standardized. For example, one papercharacterizing TCRδ and γ of I. punctatus utilized 75 %nucleotide identity in conjunction with a pairwise alignmentto define their groups (Moulana et al. 2014). In T. nigroviridis,the 13 V segments have been placed into six families based on75 % nucleotide identity (Fischer et al. 2002). Another studycharacterizing the TCRα chains in the rainbow trout(Oncorhynchus mykiss) used 75% amino acid identity to clas-sify 9 Vα segments into 6 groups and one pseudogene thatthey were unable to classify (Partula et al. 1995). In T.

rupribes, there are 17 complete V sequences that were placedin 4 subfamilies based on sequence similarity, but it was un-clear what percentage was used (Wang et al. 2001b). Furthercomplicating the family analysis is the phenomenon that TCRα and δ typically share a common pool of V segments. Themost common way to classify a V segment as either α or δ isbased on expression data, however, it is not reasonable to saythat a certain V is only an α or δ, just that it has been found tobe expressed with one or the other TCR chain or both. For thisreason, for example, the V segments of T. nigroviridis areclassified as Vα/δ since they were identified at the genomiclevel and expression data was not obtained. In contrast, the Vsegments of I. punctatus and S. salar are classified as either αor δ or both because those sequences were obtained fromcDNA expression data. This does not mean that the TCRαsequences are not expressed with TCRδ or vice versa, just thatwe do not have exhaustive data.

A phylogenetic analysis was performed using one select Vsequence from each family of D. rerio, S. salar, andI. punctatus. In addition, selected outgroup sequences

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*Gg,Mm,Hs TCRD V sequences from Gallus gallus, Mus musculus,

Homo sapiens

*Hs, Mm,Dr Ig H V sequences from Homo sapiens, Mus musculus,

Danio rerio

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Fig. 7 Vertebrate TCRαδV phylogeny. Phylogenetic analysis of selectedVα/δ sequences from D. rerio as well as selected Vδ, Vα, Vγ, Vβ, andIgH V segments from various species of teleost fish and mammals for

comparison. The neighbor joining tree was drawn using MEGA 6.0 and1000 bootstrap replications. GenBank accession numbers for selectedsequences are found in Supplemental Data 3

Immunogenetics

http://www.imgt.org/

http://www.imgt.org/

representing TCRα, β, and γ V sequences of other tele-osts, as well as TCRδ from select mammalian species, andIgH V from select teleost and mammalian species wereincluded for comparisons (Fig. 7). As expected, these se-quences showed grouping first along receptor or isotypeslines. Additionally, these V sequences appeared mostly tocluster by species and not along V family lines. There area few exceptions. The D. rerio sequences tradv3.0.1 andtradv4.0.1 group with the S. salar 25.3 sequence. D. reriosequence tradv36.2.1 cluster with S. salar V2.3,T. nigroviridis V4, V7, V10, and V12. D. rerio sequencetradv6.0.1 groups with S. salar sequence V6.1 and V3.1.In regard to the subfamilies of D. rerio tradv23, tradv30,and tradv36; families tradv23 and tradv30 cluster togetheron the phylogenet ic tree while tradv36.1.1 andtradv36.2.1 do not. Importantly, the bootstrap support ofmany of these bifurcations is low.

Expression data

Three of the identified Dδ sequences (Dδ1, Dδ4, Dδ6)were found to be expressed in the three transcripts ana-lyzed. Both Jδ1 and Jδ2 were used in these transcripts aswell, and Vδ sequences tradv23.2.1, tradv23.2.2, andtradv23.3.3 were found to be expressed. Additionally,N/P nucleotides were present at the coding joint of theV-D and D-J sequences. Through our attempts at ampli-fying the repertoire of sequences expressed in thezebrafish spleen, we were only able to obtain one uniqueclone. This single clone was further supported by PacBiosequencing which produced 440 identical sequences. Noevidence was found for constant domain allelic polymor-phism, which we have seen in some teleost fish TCR αand β including zebrafish (Criscitiello et al. 2004a, b;Kamper and McKinney 2002). Because of the sameCDR3 sequence and two V(D)J coding joints, it is mostlikely that this sequence represents an individual cloneamplified from only one of the zebrafish in the pool andnot a homogenous population of γδ thymocytes withgreatly restricted (fixed) TCRδ diversity that hone tothe spleen or peripheral blood. This is supported by ourqPCR data showing that relative upregulation of TCRδ,β and γ is low compared to alpha in spleen of immu-nized adult zebrafish, and this was the only TCRδ prod-uct we isolated. TCRδ expression data from the Atlanticsalmon showed a higher diversity in their expressionrepertoire, utilizing 13 of the available Vα/δ segmentsto produce diverse TCRδ receptors (Yazawa et al.2008b). However, this case of species-specific highly re-stricted diversity has been seen before in the axolotl(André et al. 2007) and in mouse mucosal epithelia(Itohara et al. 1990).

Conclusion

In this paper, we have provided an annotation for the completeTCR α/δ locus of the zebrafish. We found no evidence forIgHV in this locus, but did find the Varray to be inverted 3′ ofthe αC as in other teleosts offering a possible explanation forthe loss of IgH use in teleosts that appears in (at least some)sharks and amphibians. We had difficulty obtaining diversecanonical expression data for TCRδ from zebrafish spleen,suggesting that γδ T cell numbers may be especially low inthe circulating periphery of normal adult zebrafish. Perhapscertain states of immunostimulation, particular tissues, or spe-cific developmental stages will reveal more TCRδ expressionand diversity. It is hoped that this study will represent animportant first step in defining the curious expression ofTCRδ in zebrafish and the annotation and phylogenetic anal-ysis of the locus will provide a useful resource to investigatorsusing this model.

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Immunogenetics

Supplemental Figure 1: Phylogenetic tree from multiple sequence alignment of selected C sequences. Created in Mega 6.0 using the Muscle and Neighbor joining tree building methodology. Ambystoma mexicanum TCR delta (AY029365.1); Callorhinchus milii TCR delta (NW_006897485 ); Cyprinus carpio TCR delta (AB541473.2/BAI83002); Danio rerio TCR alpha (AF246178.1); Danio rerio TCR beta(NC_007128.6); Danio rerio TCR gamma (AY973943.1); Dicentratchus labrax TCR gamma (EU853841); Gallus gallus TCR delta (AF175433); Ginglymostoma cirratum TCR delta(JF507709.1); Heterodontus francisci TCR delta (U22673); Homo sapiens TCR delta (A31326); Ictalurus punctatus TCR delta (HQ913590); Ictalurus punctatus TCR gamma (DQ435303); Mus musculus TCR delta (AAA51274); Ovis aries TCR delta (AJ290095.1); Paralichthys olivaceus TCR alpha (AB053227.1); Paralichthys olivaceus TCR beta (AB081559.1); Paralichthys olivaceus TCR delta (AU050806); Paralichthys olivaceus TCR gamma (AB081560.1); Salmo salar TCR delta (EF467299); Siniperca chuatsi TCR beta (EF596786.1); Takifugu rubripes TCR delta (AB222469); Tetraodon nigroviridis TCR delta (AJ410421.1); Xenopus tropicalis TCR delta (GQ262017.1)

Heterodontus francisci TCR δ C Ginglymostoma cirratum TCR δ C

Callorhinchus milii TCR δ C Tetraodon nigroviridis TCR δ C

Paralichthys olivaceus TCR δ C

Salmo salar TCR δ C

Takifugu rubripes TCR δ C

Danio rerio TCR δ C Cyprinus carpio TCR δ

Ictalurus punctatus TCR δ C

Xenopus tropicalis TCR δ Ambystoma mexicanum TCR δ

Ovis aries TCR δ C Mus musculusTCR δ C

Homo sapiens TCR δ C Danio rerio TCR α C

Paralichthys olivaceus TCR α C Paralichthys olivaceus TCR β C

Siniperca chuatsi TCR β C

Ictalurus punctatus TCR γ C Danio rerio TCR γ C

Danio rerio TCR β C

Dicentrarchus labrax TCR γ C Paralichthys olivaceus TCR γ C

TCR alpha

Mammals

Amphibian/ Reptile

Teleost fish

TCR delta

Cartilaginous fish

TCR beta

Teleost fish

TCR gamma

< Ig Domain * * Am TCRD : THVQPS--VFVLRS--------------DDAAPCLVKDFSLKQ-AEIFM------NSSSVSETGIETSAPVLSKSGKYSA------VRVREFPVSD---HVTCAWGHGGSVVMSNDVMETQKE Cm TCRD : KPVNPK--VTMFYPAIKS---NP--PASDPAALCLADEYIPKE-LSILV--HD-INDMDLTKSATTSSSIAIIDHKLYHK------TSFLTVPQDT—-TGMRCIVQHEGKNVSAEAKYT---- Cc TCRD : -ETAPV--LSILSP-----------FKGDGPDICLAAGFFPQE-KNMTL-----TLEGNEPKSLTSSNAPLFSTTKTYYY------AGFSDNKKIN-----ECKMDGQTADKTSDTPETTDKP Dr TCRA : TEVKPN--IYQVGN------------------SCLATDFTKHN--EFNI----------TDSPFLETSAVRYSGQSYYSS------FKFSSNDCKE---TGVCSSSGGGTDLER--------- Dr TCRB : NVTEPE--VKILPPSPKELCSQNTNDKKLSTLVCVATGFYPDH-VSMSW-----KVNDQERKDNISTDHIAIQDKTTLMYNISSRMVYVSEWMDPN—-NKFTCTVRFFNGDHYINVSYTTKGQ Dr TCRD : TVNSPPAFLSVLSP-----------IKGHGSDICVAAGFFPQQ-KTMIL-----TSEDGNTVNQETSNAVLSLSSKNYYY------VGSSEKKIQE------CVMDGKTAKADK---IDKPSG Dr TCRG : SVVTPK--LSGYLNK----------ANEKPAALCQAKDMFPDL-VSFKW---E-KKSSSGGWTEVSNDQIVEHSHNEDPV------TSMVILNTPE-DNIYRCTVTHEGSKDPQQIEIKKKDE Dl TCRG : QIVKPV--VSVYPAASRA---H---LEGKISLLCVASDMFPPL-VRFSW--KRQKDNGPLEKVTSADKKQLKLKESGCTA------TILQIPQRESSTYKYYCSVQHEGETVEAQIKQEVSTL Gg TCRD : RDSIPE--VIVMKSKKHK---E---DGDTGKAACLARNFYKKN-ISLEM---------SSNEVVYEPKAPIVTSNGMYNT------IKVVKVTKQS---EVTCTARLSNGNFTANSTTPEMKA Gc TCRD : SIVKPK--LSAFYPPKSS---S---KDAVQAAVCLASDFFPKD-ISLQL------AFGDKPKANVTRPSVLKPD-GSYVA------FSFLFNPENE—-ANFVCEAVHENQHSHANITLGTKSG Hf TCRD : RPENPK--LSIFYPSLAR---SDDLDPDETAAVCLASEFTPKE-IELSV-------VWDINHKSNVTRSSILLNDGYYWS------SGFLPFPKDQKPVNVTCEAKHNGDTIVQQNIKEPTAA Hs TCRD : PHTKPS--VFVMKN--------------GTNVACLVKEFYPKD-IRINL--------VSSKKITEFDPAIVISPSGKYNA------VKLGKYEDSN---SVTCSVQHDNKTVHSTDFEVKTDS Ip TCRD : PRSLPT--LSILTSH----------DSNEKLEICLAAGFFPKE-GEVSL-----YTGDNVTPENHTVENAAMSAAGTYYY------AAFSKDGIKK------CAMKDVSLDKNDVKPTAQIPS Ip TCRG : RVKEPQ--VYVYPVSTPE-------KGEKSFLLCHARGMFPDL-VRFTW-----QAKDQGGKNVDLRGDERLEQRDEVPEVRITSMLIVGKDKAKN--NNFICTVKHDSSVKDKELPIPREED Mm TCRD : PPAKPS--VFIMKN--------------GTNVACLVKDFYPKE-VTISL--------RSSKKIVEFDPAIVISPSGKYSA------VKLGQYGDSN---SVTCSVQHNSETVHSTDFEPYANS Ov TCRD : PAASPS--VFVMKN--------------GTNVACLVKEFYPKE-VTISL--------QSSKKIIEYEPAIVVSPGGRYSA------VKLGQYNDPD---SVTCSVEHNKKTWHSSDFEPKKDI Po TCRA : DEYEPS--YYELE------------DQDTTITACLATGFSKHN-ATLEH-------DLFKNGSTNMTGAARASEESLY--------SQVALWSDAS-----QCESRTGKNESEVCADVLKK-- Po TCRB : AVKSPI--VKVFRPSSKE-CRNPIDNEREKTLVCVASDFYPDH-VSVYWQIIQLNVTSGVNVTRGVTTDEAALRKDKVYTITSRLKVSAEDWYKPE—-WNFECIVRFFNGTHDTYHKDSISGP Po TCRD : SPVKPS--LSVLSP-----------LDPGGPRVCLAAGFRPQE-VAMVL-------NGSNSIDISEEEAVASLRHNSFFF------TGFSDGAISS------CEMNDVSSSNDHE-------- Po TCRG : ---------RMYRTASRD---D---LDGKSVLLCVASNMSPPL-VHFSW--KRQKEGGPLEDLTSAQGEQLELRVPERTA------AIMVVDRDASYTYKYRCNVRHEGGPTERVVPALPPST Re TCRD : DKQEPE--LSIHYPPVHQ---SG--KTDQTATVCLLGPFYPNK-VTVNI----TLDGTTDNGSRTVLTEDVLTDKGYYRT------SGFREFSHDLQPKTIGCSAKHGDKYIETKKEITISAP Ss TCRD : IAARPT--LSILTPLHKS-------GDDDQPKVCLATGFFPST-KNMNL------ILGNNDTVQLSTTKAALSSTRTYFF------AGFSKENIKR------CEMDGNVEPPVDDKDDSTDVS Sc TCRB : ------------------------KQGKGETIVCVASEFYPDH-VSVSW-----TINGSIVREGVATDNAAL-RVGKYYRITSRLRVAAETWYNPS—-IKFTCIVTFYNVTNTDYPAHIFGET Tr TCRD : TVLSPT--LFILTPLL---------SEAPPADVCLAADFRPKE-GEMVV----------NGRVQKTSNAVLSLGHKSFFF------AGFNRTIES-------CRLHNSSVPVDREQDTDQDTD Tn TCRD : LVLQPT--LFILNPQ----------PPAEPVDVCLAAHFSPRQ-GEMGV----------NGLKQETSGAVMSHRHGSFFF------AGFNRTVKD-------CRLQESSDSVDPAGKDQGTD- Xt TCRD : ---APS--VFVLKP-----------QKGDSPTACLVKDFTPKEDVEIYM-------NSTQSNGSIISASPVVSLNGTYSA------VHVDRLGTEG----LHCQAKHKG-------------- Ig Domain >< Tm ><Cyt Am TCRD : NPSVDADISKSTEECEAPTDQLIVRS------SEKVN----ILSMTVMSLRVLFAKSLAFNLLLSATFFLL--------------------- Cm TCRD : --------------------------------PPQTS----V-------------------------------------------------- Cc TCRD : ERPTQNKCETNARTSASGFIDTG---------VPKMN----FMNLLVTGLRILLAKCVAVNVMMTVKAFVF--------------------- Dr TCRA : --------------------------------DEKVN----FLSLGIFWLRILFLKTVVFNVLVTFKAWMS--------------------- Dr TCRB : --------------------------------KVKPK----GLKLVGFGYILFLSKSFLYALVVSAMFWKLKFSMKTKEIPEE--------- Dr TCRD : NPVEDNPKKTATLECHTNTTTPPSTNND----DPKTN----SMTLLVIGLRILLAKCVAVNVMLSIKAFLF--------------------- Dr TCRG : KPSVIKPSGGPDPTCPPSTETPISQESAN---PEKKQ----SLQLFVYGYAVMLMKNVLYFIVVFIVLLKRKAGKKEERS------------ Dl TCRG : PPPTTSPSPPPSLPPSVQPSLIPSPPPSLIPAPPASAPSQYQVKLLCLMYTVLMVKSLVYCCGLSCLMIFRNKGPSTNCTHAD--------- Gg TCRD : EEIKPANICNTTDTSAEDVK------------MEKVN----MLSMAVLGLRVLLAKSIAFNTLMSIKLILF--------------------- Gc TCRD : TGPTQSSCAESSKDMMEAGNDPFQE-------RPQAN----LLSLTLMGLRFLLFKSISVNLLMTARVWIS--------------------- Hf TCRD : PPKPIDCNKSSNGTSAGLNDTDND--------LTEWN----FMSLTVMGLRVLFFKSVAFNVMMTARCCSFKEFSAMRWIQR---------- Hs TCRD : TDHVKPKETENTKQPSKSCHKPKAIVH-----TEKVN----MMSLTVLGLRMLFAKTVAVNFLLTAKLFFL--------------------- Ip TCRD : CDQNSTLGISTSNNLTSPNKIKISG-------DPKGN----TMLLIVTGLRLLLAKAVGINILMTIKAFLV--------------------- Ip TCRG : TSKSNAGVLNTCPTQKAEEAEEVEEEIMNFGVFEHSR----SLYLFSVTYVLLLVKNVLYFCTVFVLLFKRNPAKI---------------- Mm TCRD : FNNEKLPE------------------------PENDT----QISEPCYGPRV-----TVHTEKVNMMS------------------------ Ov TCRD : SETTPKPTESENTTEIQVPATCYEPQVQ----PGKVN----MMSLSVLGLRMLFAKSVAVNFLFTAKLF----------------------- Po TCRA : --------------------------------DPAVN----TVSLLVVALRLLFLKTVVFNVLLTLRLWLSHRV------------------ Po TCRB : DILTR---------------------------EKYLR----ITRQAKLSYSVLIIKSSVYGAFVAFLVWRLQSSTEKQN------------- Po TCRD : --------------------------------NVRFN----SYLLLLNAARVMFTKIVAFTSVLTIRTLLA--------------------- Po TCRG : PPPSTTPPSTPPPSETSP--------------EPTWS—QQQLKLLCLLYTVLMVKSLVYCCGLSLINILRNRERPPAAHKRTELPPAASTSH Re TCRD : GVTPALDCDAGTSSNHTGTD------------MPEVN----FMTLTVMGLRILFFKSVAFNVLMTARAWVF--------------------- Ss TCRD : NYNTTATTASVNCKDEPLNNVTTTNYTD----YTKMN----FTSLVVYGLRVLFAKAVAFNVLFTVKALVF--------------------- Sc TCRB : AVGDGMTR------------------------DKYLR----ITQTAKLSYGVFIIKSCIYGAFVVFLVWRLQGSCGKQNN------------ Tr TCRD : QDTDQDTDQDTDQDTDQDTVQDTDQDTDQEWT-------RSREN----LLLLRMDMLRVVLTKTVSLSTIVTIRALLL-------------- Tn TCRD : --------------------------------PELWT----VTRRNLLLLQ----------------------------------------- Xt TCRD : --------------------------------------------------------------------------------------------

Supplemental Figure 2: Multiple sequence alignment of selected C sequences. Created in Mega 6.0 using the Clustal W methodology. Ambystoma mexicanum TCR delta (AY029365.1); Callorhinchus milii TCR delta (NW_006897485 ); Cyprinus carpio TCR delta (AB541473.2/BAI83002); Danio rerio TCR alpha (AF246178.1); Danio rerio TCR beta(NC_007128.6); Danio rerio TCR gamma (AY973943.1); Dicentratchus labrax TCR gamma (EU853841); Gallus gallus TCR delta (AF175433); Ginglymostoma cirratum TCR delta(JF507709.1); Heterodontus francisci TCR delta (U22673); Homo sapiens TCR delta (A31326); Ictalurus punctatus TCR delta (HQ913590); Ictalurus punctatus TCR gamma (DQ435303); Mus musculus TCR delta (AAA51274); Ovis aries TCR delta (AJ290095.1); Paralichthys olivaceus TCR alpha (AB053227.1); Paralichthys olivaceus TCR beta (AB081559.1); Paralichthys olivaceus TCR delta (AU050806); Paralichthys olivaceus TCR gamma (AB081560.1); Salmo salar TCR delta (EF467299); Siniperca chuatsi TCR beta (EF596786.1); Takifugu rubripes TCR delta (AB222469); Tetraodon nigroviridis TCR delta (AJ410421.1); Xenopus tropicalis TCR delta (GQ262017.1).

V12.0.1 ---------ATTATACTCATGGAGAAGGAATAGCTCCTCT------CTCTTCATCTGAATTGGTCATCAATGGTCACTCCACAACTTTGTCCTGTAATTACAATGGCTCT---TACAGCAGTGACT-------CTCTCATGTGGTA--TCGACAATACAG---CAGCTCCAAACCACAGTTTCTGTATTT V12.0.2 ---------ATGATACTAGTGGAGAAGGAATAGCTCATCA------CTCTTCATCTGAATTTTTCATCAATGGTCACTCCACAACTTTGTCCAGCAATTACAATGGCTCT---TACAGCAGTGACT-------CTCTCTTGTGGTA--TTGACAATACAG---CAGCTCCAAACCACAGTTTCTGTATTT V13.0 ---------GTGATGCCTTTGGTGAGGGAATTGCACCTGT------CTCGGGTTACAAAACCGTCCCAGAGGAGAAAACAACGACCCTGTCCTGCATTTATAATGGCTCA---GCAGCTACAGATG-------CTCTCCAGTGGTA--TCGTCAGTACCC---AAGATCCAGACCAGACTTCCTTTTTCT V5.0.1 ---------GAGACTCTCTTGGTGACATTATAACATCTCG------TGGTTCTGAAAAAGCAGCTCAAGATGGTACCAATGTCACTCTGTCCTGCAACTACACTGGTGC---------GGTTTATA-------ATCTGTTATGGTA--TCGTCAGTATCA---GAGATTCAAACCTGAACTCCTTCTCTC V5.0.2 ---------GAGAAGTAATAGGTGAAGTGATAACATCTCG------TGGTTCTAAAAAAGAAGCTCAAGATGGTACCAAGGTTACTCTGTCCTGCAACTACACAGGAGT---------GGTTTATA-------ATCTGTTATGGTA--TCGTCAGTATCA---GAGATTCAAACCTGAACTCCTTCTCTC V17.0 ---GCACCAGCAGACAAGACAGCATTACTCCAACATCTCC------TGCTGTCCATGTAAAAGAAAAGGAAGCAGCCAGAATTTCTTGTGAATACAAGTACACAGTGTC---------CATGAATA-------ATCTGCAGTGGTA--TCGCCAATATCC---CAATGCTAAGCCAGAATTCCTGGTTCT V3.0.1 ---------GGAATGCTGGTAAGGAAGTGATATCATCACT------GTCCACAGAACAGATTGTTTTGGCTGGAAAAAATGCCACACTTTCCTGCAAATACAATAG---------CAATATTCGCA-------ATTTGCAGTGGTA--TCGACAGTATCC---CGGATCTAAACCAGAGAACATCATTTT V3.0.3 ---------AGACTGCTGGTGAGGAAGTGATATCATCACT------GTCCACAGAACAGATTGTTTTGGCCGGAAAAAATGCCACACTTTCCTGCAAATACAATGG---------CAATGTTTACA-------GCTTGCAGTGGTA--TCGACAGTATCC---CGGATCCAAACCTGAGAATATCATTTA V3.0.2 ---------GGGATGCTGGTGAGGAAGTGATATCATCACT------GTCCACAGAACAGATTGTTTTGGCTGGAAAAAATGCCACACTTTCCTGCAAATACAATGG---------CAATGTTTACA-------ATTTGCAGTGGTA--TCGACAGTATCC---CGGATCCAAACCTGAGAATATCATTTT V3.0.4 ---------AGAATTCGGGTGAAGAAGTGATATCAACACT------ACCCACAAACCAATATATTATTGAAGAAAAAAATGCCACACTTTCCTGCAATTACAGTGG---------CTATGTTCAAA-------ATTTGCAGTGGTA--TCGTCAGTATCC---TGGATCCAAACCTGAGAATATTATTTT V4.0.1 -----------AATGCTGGTGAAGAAGTGATAAGACCACT------CTTCAAAGAGAAACATGTTCTGGAAGGAGATGTTGTTACACTGATCTGCAGCTACTCTGG---------AAGTGTTCAGA-------ATCTGCAGTGGTA--CCATCAGTTTCC---CGGATCTAAACCTGAAAATATATTTCT V4.0.3 ---------AGAATGCTGGTGAAGAAGTGTTAAGACCACT------CTTCAAAGAAAAACATGTTCTGGAAGGAGATGTTGTTACACTGATCTGCAGCTACTCTGG---------AAATGTTCTGA-------ATTTGCAGTTGTA--CCATCAGTTTCC---CGGATCTAAACCTGAAAATATATTTCT V4.0.2 -------------TCATTACAGTGGA-CATTATTTATACT------GTTCAAAGAGAAACATGTTCTGGAAGGAGATGTTGTTACACTGATCTGCAGCTACTCTGG---------AAATGTTCAGA-------ATTTGCAGTGGTA--CCATCAGTTTCC---CGGATCCAAACCTGAAAATATATTTCT V9.0.1 --------AAAGCGTTTTTTTCAAATGCAATAAGACCTCT------CTCTGCTGAACAGCAGGTTTTAGAAGGAGGAAAGGTGATCCTTTCATGTGCCTACAATGGAAG------TAATATAAAGA-------GTTTACAGTGGTA--CAAACAGCATC---ACAGCCCTGCACCCGAATATCTCCTGCA V9.0.2 --------TAGGCATTTTTG-CAAATTCAGTAACTCCTTG------GTCAACTAAACAGCAGGTTTTGGAAGGAGAAAATGTGACTCTTTCATGCAACTACAGTGGAAG------TAATATTCAAA-------GTTTGCAGTGGTA—CAGAAAGTATCCAAGCAGCACAGCTCCTGAATATATCCTGCAA V8.0 ---------AGATTGCTGATGCATATACAATAGAATCTCT------CTCAGCTGAAAAGCAGGTTTCAGTTGGAGGAAATGCTGTGATTTCTTGTAAATACAGTGG---------TATTGGGCCAA-------GCTTCCAGTGGTA--TCGCCAATATCCA---GGATCTAGACCAGAGTACCTTATTTT V6.0.1 ---------AACACACAGAGGCAGATGTAATAAAACCTTT------ATCCCTGGATGAAAATAGCCATGTTGGTGATGCTGTTACTCTGTCCTGCAGTTACAAGGATTATACGGGTAATATGCAGA-------ATTTACAATGGTA--CCGGCAATTTCC---CAGATCCAAACCAGAGTTCATCCTTTA V6.0.3 ------CACAACACACAGAGGCAGATGTAATAAAACCTTT------ATCCCTGGATGAAAATAAACATGTTGATGACACCTTTACTCTGTCCTGCAGTTATAAAGATTATACGGACACTGTACGGA-------ATTTACAATGGTA--CCGGCAATTACC---CAGATCCAAACCAGAGTTCATCCTTTA V6.0.2 ---------AACACACAGAGGCAGATGTAATAAAACCTTT------ATCCCTGGATAAAAACTGTCATGTTGGTGACGCTGTTACTCTGTCCTGCAGTTATAAAGATTATACGGGTACTGTACAGA-------ATTTACAATGGTA--CCGGCAATTTCC---CAGATCCAAACCAGAGTTCATCCTTTA V6.0.4 ---------AACACACAGAGGCAGATGTAATAAAACCTTT------ATCCCTGGATGAAAACAGTCATGTTGGTGATGCTGTTACTCTGTCCTGCAGTTATAAAGATTATACGGGTACTGTTAATG-------GTTTACAATGGTA--CCGACAATTACC---CAGATCCAAACCAGAGTTCATCCTTTA V7.0 -----AATGTTGATGCAGATGGACATATGATAAATCCTTT------GAGTTTAGAGACACACGGCCCAGTAGGACACAATGTGACCCTCTCCTGCAATTACAGTGGTCGT---------GTTCATA-------CTCTGCAATGGTT--TCGACAGTATCC---AGGATCTCGAATAGAGTTCCTCATTTT V34.0.1 ---------GAATGTCTTTTTCAGATGAAATACATCCCCT------GTCTTCAGATAAACATGTAGCTGAAGGTCAAAATGTGACTCTATGTTGTAATTACAGAGCCAGTACTGGCAATGTTGACT-------CTCTCCTGTGGTA--TCGTCAGTATCC---CGGAGCAAACCCAGAGTATTTACTGCT V34.0.3 ---------GAATTTCAATTGCAGATGAAATACATCCCCT------GTCTACAGACAAACATGTAGCTGAAGGTCAAAATGTGACTCTGTCTTGTAATTACAGCACCAGTACTGGCACTGTTAACT-------CTCTCCAGTGGTA--TCGCCAGTATCC---AGGAGCAAAACCAGAGTTTTTACTTTC V34.0.4 ---------GAACTGCAATTGCAGATGAAATACAGCCCCT------GTCTTCAGACAAACGTGTAGCTAAAGGCGAAAATGTGACTCTGTCCTGTAATTACAGCACCA---CTGTCACTGTTGACT-------CTCTCCAGTGGTA--TCGTCAGTATTC---CGGAGCCAAACCTGAGTTTTTACTTTC V34.0.2 ---------GAACATCTTCTGCTGATGAAATACATCCCCT------GTTTTTAGATAAACAGGTAGCCGAAGGTCAAAATGTGACTCTGTCTTGTAATTACAGCATCAGTATTGGCATTCTTAGGT-------CTCTCCAGTGGTA--CCGCCAGTATTC---CGGAGCAAAACCTGAATTTTTACTGAT V34.0.5 ---------GAAATTTATTTGGTGATACAATCGAACCGCT------GTTTCCAGACAGGCATGGTTCTGAGGGTGAAAATGTGACTTTCTCCTGTAACTACAGTCTCACTAGTGGCACCAGTGTTA-------ATCTTCTGTGGTA--TCGCCAGTATTC---TGGAGCAAAACCAGAGTATTTACTTTC V34.0.6 ---------GAAATTCTTTTGGGGATGAAATTCAATCCCT------GGTTAAACACGAACATGGTTCTGAGAATGAAAATGTGACTTTCTCCTGTAAATACCGTCTAACTAGTGGGACTAATGTCA-------ATTTCCAGAGGTA--TCGCCAGTATTC---CGGAGCAAAACCAGAGTATTTACTTTC V10.0.2 ------------ATACACTGGCACAGTCAATAAGACCACT------GCAGAGCGATGTTCATATCACTGAGAAGACTCCAGTCAGGCTCTCATGCAAATATGATGG---------CAGTGCAAACA-------GTCTGCATTGGTA--TAGACAGCACCT---CGGATCAAAACCTGAATTTCTGCTTCT V10.0.3 ------------ATACACTGGCACAGTCAATAAGACCACT------GCAAGGCGATGTTCATATCACGGAGAGGACTCCAGTCAGGCTCTCATGCAAATATGATGG---------CAGTGCATACA-------GTCTGCATTGGTA--TCGACAGTATCC---AGGAGCAAAACCTGAATTTCTGCTTCT V10.0.1 ------------ATACACTGGCACAGTCAATAAGACCACT------GCAGAGCGATGTTTATATCACTGAGAGGACTCCAGTCAAGCTCTCATGCAAATATGATGG---------CAGTGTATACA-------GTCTGCATTGGTA--TCCACAGTATCC---AGGAGCAAAACCTGAATTTCTGCTTCT V10.0.4 ------------ATACACTGGCACAGTCAATAAGACCACT------GCAGAGCGATGTTTATATCACTGAGAGGACTCCAGTCAAGCTCTCATGCAAATATGATGG---------CAGTGTATACA-------GTCTGCATTGGTA--TCGCCAGTATTC---AGGAGCAAAACCTGAATTTCTGCTTCT V10.0.5 --------ACGAATGC-TTTGCGCAAAATATCACACCACT------GCAGGGCACGACACAAGTTACTGAGGGAAAGACAGTCACTCTGTCATGTAAATATGATGT---------CAGTGTCCAGA-------GTTTGCTTTGGTA--TCGACAGTATCC---TGGATCAGGACTGGAATTTCTGCTGCT V38.0.1 ---------AGGAGACGTTTGCACAATCAATAACACCACT------GGAAAACAAGACACTTAAAAGTGAGGGAGAAACGGTCACCCTGTCCTGTAAATATGAAGG---------CGTTGTGAATA-------ACCTGCACTGGTA--TCGTCAGTATCC---TGGATTCAGACCAAAGTTCCT------ V38.0.2 ---------AAGAGACGTTTGCACAATCAATAACACCACT------GGAAAACAAGACACTTAAAAGTGAGGGAGAAACGGTCACCCTGTCCTGTAAATATGAAGG---------CGTTGTGAATA-------ACCTGCACTGGTA--TCGTCAGTATCC---TGGATTCAGACCAGAGTTCCT------ V38.0.3 ---------GTGACACATTTGCACAGACAATAACACCGCT------GGAAAACAAGATAGTTAAATCTGAGGGAGAAACTGCCTCTCTGTCCTGTAAATATGAAGG---------GTCAGTAACTA-------ACTTGCACTGGTA--TCGTCAGTATCC---TGGATCCAGACCAGAGTTCCT------ V11.0.1 ---------ATCAATCATTTGGCCAGTCAATAACAGCACT------GCAGAGCACAAAACAGGCCGCTGAGAGCGAAACGGTCATTCTGTCATGCAAATATGATAC---------CCGAGCAGACA-------ATTTACAGTGGTA--TAGACATTATCC---AGGATCTACACCAGAATATCTGCTGCT V11.0.2 ---------ATCAATCATTTGGTCAGTCAATAACAGCACT------GCAGAGCACAAAACAGGCTGCTGAGAGCGAAACGGTCAGTCTGTCCTGCAAATATGATAC---------TCAAGCAAACA-------ATTTACAGTGGTA--TCGACAATATCC---AGGATCTACACCAGAATATCTGCTGCT V11.0.3 ---------ATCAATCATTTGGCCAGTCAATAACAGCACT------GCAAACCACAAAACAGGCTGTTGAGAGCGAAACGGTCATTTTGTCCTGCAAATATGATAC---------TCAAGCAGACA-------GTTTACACTGGTA--TCGACAATATCC---AGGATCTACACCAGAATATCTGCTGCT V14.0 ---------GTATCATCCAGACAAACATGATAACATCCCA------TGACCCACAAAAACAAGCATTTGAAGGGGAATATGTAAAACTTGCATGTAACTACAGTGG---------AGCACCTGATA-------ATCTTCACTGGTA--CCGTCAATATCC---CGGATCGACACCAATGTTCCTCCTTTA V36.2.8 -----ACCGGCTGTTT--TTGGAAATGTCATCAAACCAAA------GCAAACTGATGTTTTTGCAGATGAAGAATCAAATGTGACATTATCCTGTAGTTTTACAGCCTCTGG---AGGGTCAGATT-------ATCTCCACTGGTA--CCGTCAGTATGG---AAGATCAAAACCTGAATTTCTTGTACT V36.2.10 -------CGGCTGTTT--TTGGAAATGTCATCAAACCAAT------CAACACTGATGTTTTTGCTGAGGAAGGATCAAATGTGACATTATCCTGCAGTTTTACAGCCTCGGG---TGTCACAGATT-------ATCTCCACTGGTA--CCGTCAGTATGG---AAGGTCAAAACCTGAATTTCTTGTAC V36.2.2 -------CTGCTGTTT--TTGGAAATGTCATCAAACCAAT------CAACACAGATGTTTTTACCGAGGAAAGATCAAATGTGACATTATCCTGTAGTTTTACAGCCTCTGG---AGGGTCAGATT-------ATCTCCACTGGTA--CCGTCAGTATGG---AGGATCAAAACCTGAATTTCTTGTCCT V36.2.11 TTTTCAGCTGCTGTTT--TTGGAAATGTCATCAGACCAAT------CAACACTGATGTTTTTGCTGAGGAAGGATCAAATGTGACATTATCCTGTAGTTTTACAGCCTCTGG---AGGTACAGATT-------ATCTCCACTGGTA--CCGTCAGTATGG---AGGATCAAAACCCGAATTTCTTGTACT V36.2.9 --ACACCTCACTAAAAAATCAAATGTGTCTTGGATCTACTGGTATGTAAAACAGATGTTTTTGTTGAGGAAGGATCAAATGTGACATTATCCTGTAGTTTTACAGCCTCTGG---AGGGTCAGATG-------ATCTCCACTGGTA--CCGTCAGTATGG---AGGATCAAAACCTGAATTTCTTGTACT V36.2.1 -------CTTCTGTTT--ATGGAAATGTCATCATACCAGA------TGAAACAGAAGCTGTTCATGCGAAGGGTTCAGATGTTAAACTATCCTGCAGTTTTTTAGCCTCTGG---ATATACAGACT-------ATGTCTACTGGTA--CCGTCAGTATAG---AAGATCAAAACCTGAATATCTTGTACA V36.2.12 -------CAACTGTCT--TTGGAAATACCATCAAACCAGA------TGAAACAAATGTAGTTGCTCAGAAGGATTCAGATGTTAAATTGTCCTGTAGTTATTCA---TCTAC---AACTACAGATA-------CTCTCTACTGGTA--CCGTCAGTATGA---AAGATCAAAACCAGAATTTCTTGTACT V36.2.6 -------CTTCAGTCT--TTGGAAGTGATATCAAATCAAA------CATCACAGATGTTGTTTATAAGGAGGGTTCAGATGGTACAATATTCTGCAGTCATTAT---CCTGG---AACTGGAGATA-------ATATCTTCTGGTA--CCGCCAATATGG---AGGATCAAAACCTGAATTTTTGCTTGC V36.2.7 -------CTTCAGTCT--TTGGAAATGATATCAAACCAAA------CTCAACAGATGTT---TATGAGGAGGGTTCAGATGGTACAATATTCTGCAGTCATTCT---CCTGG---AGCTGGAGATT-------ATATCTACTGGTA--CCGTCAGTATGG---AGGATCAAAACCTGAATTTCTTGTATT V36.2.5 -------CATCTGTTC--TTGGAAATGACATCAAGCCAAA------CATCACAGATGTTGTTTATGAGGAGGGTTCAGATGGTACAATATTCTGCAGTCATTCT---CCTGG---AGCTGGAGATT-------ATATTTACTGGTA--CCGTCAGTATGG---AGGATCAAAACCTGAATTTCTTGTACT V36.2.4 -------CTTCAGTCT--TTGGAAATGATATCAAACCAAA------CTCAACAGAAGTTGTTTTAAAGGAGGGTTCAGATGGTACAATATTCTGCAGTCATTCT---CCTGG---AACTGGAGATT-------ATATTCACTGGTA--CCGTCAGTATGG---AGGATCAAAACCTGAATTTCTAGTTCT V36.2.3 -------TTTCGGTTT--CTGGAAACCTCATCAAACCAAT------CCAAACTGATGTTTTTGCTGATGAGGGTTCAAATGTGACATTATCCTGCAGTTATTCA---TCTG---------CAGATT-------ATCTATTCTGGTA--TCGTCAGTATCC---CAGAGGAGCTCCTGAATTTCTTGTGTC V25.0.2 -------GTGTCAGTT--TTGGGAATGTCATCACATCGGT------TGAAACCCGTCTGCTTCATAGCGAAGGAGACGTTATAAGTGTCTCCTGTAGCTACTCC---TCTG---------CAAGAA-------CTTTATTCTGGTA--TCGCCAAAATCC---TGGAACGGTTCCTCAATTTCTTTTTGT V25.0.3 -------GTGTCAGTT--TTGGGAATGTCATCACATCGGT------TGAAACCCGTCTGATTCATACAGAAGGAGACGTTATAAGTGTCTCCTGCAGCTACTCC---TCAG---------CAAGAA-------CTTTATTCTGGTA--TTTCCAAAATCC---TGGATCGGTTCCTCAATTTCTTTTCAT V25.0.1 -------GTGATGTTT--TAGGAAATGTCATCATACCGGT------CCACTCTGAAGTATATAAAACAGAAAAGGAAACTATATCTCTGTCCTGCAACTACTCT---TCAG---------CATCGA-------GTCTCTACTGGTA--TCGTCAATATCC---TAAATCAACACCTGAATTTCTTTTTGT V25.0.4 -------GTGTTACTT--TTGGGAATGACATCACACCAGT------CCAGACCGAGGTGTTTGGGGCAGAGATGGACAATATAACAGTGTCCTGCAAATACTCT---TCAG---------CAGTTA-------GTCTGCAGTGGTA--TCGCCAGTATCC---TGGTTCAGCACCTGACTTTCTTTTGAC V15.0 -------GTGTCAGCT--GTGGCGATGTCATCACTCCAGC------CCAAACTGAGGTGTATGGGAAAAGTGGGAATAAAGTCACGCTGTCGTGCAACTACTCC---ACAG---------CACTTT-------CTTTATACTGGTA--TCGTCAATATCC---AGGATCAAATCCACATTGCCTTCTTCT V19.0 -------GTGGAAAGT--CTGATAGTCCCATCACATCAGA------TCAGACTGAGGTCTTTCCTAGGGAGAATGCCAATGTCACTTTGTCCTGCAGTTACTCA---TCAG---------CAATCA-------GTCTGCATTGGTA--CCGCCATTTACC---CAGATCAGCACCTGAATTTCTTCTATT V20.0 -------ATGTCACTC--ATGCAGATGCAATTACCCCAGA------CCGCCCTGAAGTGAATGTTAATGAGACCGAAAGCATAACGTTATCCTGTCAATATTCC---TCAG---------CATTCA-------GTCTCCACTGGTA--TCGACAGCATGA---TGCATCCGGTCCTCAGTTCCTCATGCT V37.0 -------GTGTCATTT--TTGGAGATGAAATCAGTCCTGA------CAGCAGTGAAGTGTTTGCTGCAGTGGCTTCAAAAGTAACTTTATCCTGCAGGTATTCC---TCAG---------CATATA-------ATCTTCAATGGTA--CCGTCACTATCC---TGGATCAGCTCCTCAAAATCTTGTCCT V36.1.2 -------ATGCAACAT--ATGCAGATGAAATTAAACCAGT------CAAAACTGAGGTGTTTGCTGATGAGGGCTCAAATGTTACTTTATCCTGCACTTATTCA---TCTG---------CAAATA-------ATCTTCAATGGTA--CCGTCAGTATCC---TGGATCAGCTCCACAATATCTTGTGCT V36.1.3 -------ATGCAATAT--ATGCAGATGAAATTAAACCAGT------CAAATCAGAGGAGTTTGCTGATGGGGGCTCAAATGTTACTTTATCCTGCACTTATTCA---TCTG---------CATATT-------ATCTTTACTGGTA--CAGACAGTATCC---TGGATCAGCTCCTCAACATATTGTGCT V36.1.1 -------ATGCAACAT--ATGGAGATGAAATTAAACCAGT------AAAAACAGAGAAGTTTGCTGACAAGGGTTCAAGCGTTAAATTATCCTGCACTTATTCA---TCTG---------CAAATA-------CTCTTTTCTGGTA--CCGTCAGTATCC---CGGATCAGCTCCTCAATATCTTGTGCT V36.1.6 -------GTGTTGCAT--ATGGAAATGAAATTAAACCAGT------CAAAACAGAGGAGTTTGCTAAGGAGGATTCAAGTGTTACTTTATCCTGCAGTTATTCA---TCTG---------CACGGA-------CTTTATTCTGGTA--CCGTCAGTATCC---TGGATTAGCTCCTCAATTCCTTGTGTT V36.1.4 -------ATGCCAGTA--ACGGAGATACTATTACACCAGA------CAAAACAGAGGAGTTTGCTGATGAGGATTCAAATCTTACTTTATCCTGCAGTTATTCC---TCTG---------CCTGGA-------ATTTACTTTGGTA--CCGCCAGTATCC---TGGATCAGCTCTTGAATATCTTGTGCT V36.1.5 -------GTGCCAGTA--GTGAAGATACTATTACAGCAGA------CAAAACAGAGGAGTTTGCTGATGATGGTTCAAATGTTACTTTATCCTGCACTTTTTCA---TCTG---------CATGGT-------CTTTACTTTGGTA--CCGTCAGTATCC---TGGATCAGCTCCCGAGTTCCTTGTAAA V24.0.1 ---------GTAACCTTGCTAAAGATGTGATCACACCATA------CAGTGATGAAATTTTTGCCAGCGAGACTGAGAGTGTGAAATTCTCCTGTAACTACACTGGTTCTG---------TTGACG-------GTCTTCACTGGTA--TCGGCAGTATCC---AGATTCACCACCACACTTTCTCATACT V24.0.2 ---------GTAACCGTGCTAAAGATGCGATCACTCCATA------TAGGGATGCAGTTTTTGCCAGTGAGAGTGAGAGAGTGAAACTCTCCTGTAACTACACTGGTTCTG---------TTGATA-------GTCTTCACTGGTA--TCGACAGTATCC---CGGTTCACGACCACACTTCCTCATACT V24.0.4 ---------ATTCCAGTGCTACAGAAGCCATCACACCATA------CAGGGCTTATGAATTTGCTTTTAAGGATGACAGAGTGATCCTCTCCTGTAACTACACTGGTTCTG---------CTCGAG-------GTCTGCACTGGTA--TCGACAGTATCC---AGGTTTACCACCCCATTTCCTCATACT V24.0.3 ---------GTTCCAGAGCCTCGGAAGTCATCACTCCTAA------AACTGACAGAGAGTTTGCTGTTGAAGGTGACAATGTGACTCTCTCTTGTAACTACAGTGGATCAG---------TTCGTA-------GTGTTCACTGGTA--TCGCAAATATTC---AGGTTCACCTCCACAGTTCCTCATACT V24.0.5 ---------GTTCCAGAGCTTTAGAAGCCATCGATCCAAA------AACTGACAGAGAGTTTGCTGTTGAAGGTGACAATGTGACTCTCTCTTGTAACTACAGTGGATCTG---------TTCGTG-------GCGTTCACTGGTA--TCGACAGTACGC---AGCTTCGCCACCACAATTCCTCATACT V28.0 ------GTGTTTCTTCAGCTGAAGAAATCATATTCCCTAA------CAATAAAACAGTAATTGCCAAAGCAGATGAAAAAGTTCTCCTATCCTGCAGCTACACTGGCACGG---------TTTATG-------ATTTATATTGGTA--CCGTCAATCCCC---TGGATCAGCACCTCAGTTCCTCATAAT V2.0.1 ---------GTGTAGTAAATGGGAATGTCATCACACCAGC------GAGAACATCTGTAGTTTTAACTGAAGGAAGCAACATCACTCTGTCTTGTACATTTAATACATCGC---------CTTACA-------GGCTCCTCTGGTA--CCGACAGACGCC---TGGATCAAGACCAGAGTTTCTGCTGCT V2.0.3 ---------AGGACTCAAATGGGAATGTCATCAGACCAGA------GAAAACATCTGTAGTTTTAACTGAAGGAAGCAACATCACTCTGTCTTGTACATTTAATATAACAC---------CTTACA-------GCCTCCACTGGTA--CCGCCAGATGCC—TGGATCAAGACCAGAGTTTCTGCTACTGA V2.0.4 ---------GAAACGCTAATGAGAACTCTATCAGACCAGA------TGAAGAGTTTGTGTCTTTAAACGAAGGCAGTAACACCACCCTGTCCTGTACATTTGATGGATCTG---------CTTATC-------GCCTCCACTGGTA--TCGACAGAAAAC---TGGATCGTTGTTAGAGTTTCTAGTGCT V2.0.2 ---------GTGACTCTAATGGTGATATTATTAGACCAGA------GCAAACATCAGTAGTTGTAACTGACGGAAGCAGCATCACTCTGTCCTGCACATATGATGAATCAG---------CCTACA-------GTCTCCACTGGTA--CCGACAGACACC---TGGATCAAGACCAGAGTTTCTGCTGCT V1.0 ---------GTGAAACAACTGGGGATAAAATTAAACCAGA------TAACGACACCGCACAGTTAATGGAGAATTCAAATATAACACTCTCATGCACATATGAAGGCTCCA---------TTGACA-------GCTTGTACTGGTT--CCAACAAAAAAT---TGGATCAAGACTGGAGTTTCTCATTAT V23.2.6 -------GTGTGACGAATGCA--GATCAGATTGGACCAAATAAGGGTGAAAAT---TCCATCAGTAAAGAGGGAGAGACTG----------------------------------------ATATG----TTAGCCTTTACTGGTATACAGACAATATAC---TAACAAAGAACTTCAATATTTAATATG V23.2.4 -------GTGTGACGAATGCA--GATCAGATTGGACCAAATAAGGGTGAAAAT---GCCATCAGTAAAGAGGGAGAGACTGCTACAATGAGCTGCTCGTATGATACA------AGCAGCAGATATG----TTAGGCTTTACTGGTA--CAGACAATATAC---TAACAAAGAACTTCAATATTTAATATG V23.2.8 -----AGGTGTGACGAATGCA--GATCAGATTGGATCAAATATTGGTGAAAAT---GTCTTCAGTATAGAAGGAGAGACTGCTACATTGAACTGTTCATATGCTACA------AGCAGCAGGAATG----TTAGACTTTATTGGTT--CAGACAATATCA---TAATAAAGAACCTCAATATTTAATATG

V23.2.7 -------GTTTGATGAGTGTG--GATAAAATTGGGCCAGATAAAGACACAAAT---GTCATCAGTAAAGAAGGAGAGACTGTGACACTAAGCTGTTCATATGATACA------CAGAGCAGCTATG----TTAGACTTTACTGGTA--CAGACAATATGC---TGACAGAGAACCGCAGTATTTAATTTG V23.2.1 -------GTGTGATGACTGCA--GACCAGATTAGGCCAAATAAAGAAGC---T---TTTACTGTAAAGGAAGAGGAGACTGTGACCTTCAGTTGCTCATATGAAACA------AGCAGCAGCTATG----TTTGGCTTTACTGGTA--CAGACAGCATCT---TAACGGAGAACCTCAGTATTTAATCTT V23.2.2 -------GTGTGATGACTGCA--GACCAGATTAGCCCAAATAAAGAAGC---T---CTTACTGTAAAGGAAGAGGAGACAGTGACCTTCAGTTGCTCATATGATACA------AGCAGCAGTTATG----TTAGGCTTTACTGGTA--CAGACAATATCT---TAATGGAGAACCTCAGTATTTATTATT V23.2.3 -------GTGTGATGACTGCA--GACCAGATTAGGCCGAATAAAGATGT---C---ACAGTCATAAAGGAAGAGGAGACTGTGACCCTCAGCTGCTCATATGA---A------AGCAGCAGTACTG----TTAGGCTTTACTGGTA--CAGACAGTATCT---CAATGGAGAACCTCAGTTTTTAGTATC V23.2.5 -------GTGTGATGACTGCA--GACCAGATTAGGCCAAATAAAGATTC---C---ACAGTCATAAAGGAAGAGGAGACTGTGACCCTCAGCTGCTCGTATGA---A------AGCAGCAATTATG----TTAGGCTTTACTGGTA--CAGACAGTATCT---CAATGGAGAACCTCAGTATTTAATACA V40.0 -------ATGTGACGGCTGTG--GACAAAATTGAGCCAAACAGTGGGAGCAAA---GTCATCAGAACAGAACGAGAGTCTGTAAAGCTGAGCTGCTCTTATGATTCA------AATAATGAATATG----TACGTCTATATTGGTA--CAGAAGACATTC---AAGTGGAGAACTTCAATATTTATTATA V23.3.4 -------GTTTGGTGTGTGAA--GACAGCATTGAGCCAGATAAAGGGACAGAA---AAAACATCTGAAGAAACAGAAAATATTAAGCTAAGCTGCTTATATACTACA------AGCAGTGATTATG----TTTGGCTTTACTGGTT--CAGACAATATCT---CAAAGGAGAACCTCAGTATTTATTATA V23.3.7 -------GTTTGGTGTGTGGA--GACAGCATTGAGCCAGATAAAGGGACAGAA---AAAAACTCTGAAGAAACAGAAAATGTCAAGCTGAGCTGCTCATATAGTACA------AGCAGTGAATATG----TTTATCTTTACTGGTA--CAGACAGTATCT---CACTGGAGAACTGCAGTATCTACTACG V23.3.6 -------GTTTGGTGTGTGGA--GACAGCATTGGGCCAGATAAAGGAACAGAA---AAAA---CTGAAGAAACTGAAACTGTCAAGTTGAGATGCTCATATAGTACA------AATAGTGAATATG----TTTGGCTATATTGGTA--CAAACGGTCTCT---CAATAAAGAACCTCAGTATTTATTGTT V23.3.5 -------GTTTGGTGTGTGGA--GACAGCATTGAGCCAGATAAAGGGACAAAA---AAAAAGTCTGAAGAAACAGAAAGTGTCAAGTTGAGCTGCTCTTACAGTACA------AGTGGTGAAGATG----TTCTGCTTTACTGGTA--CAGACAGTATCT---CAATGGTGAAACTCAGTATTTACTGCG V23.3.1 -------GTTTGGTGTGTGGA--GACAGCATTGAACCAGATAAAGGGAAAGAA---AAAACATCTGAAGAAATAGAAAATGTCAAGTTGAGCTGCTCTTACAGTACA------AGCAGCAGTTATG----TTTGCCTCTACTGGTA--CAGACAGTATCT---CAATGGAGAACCTCAGTATTTAGTGCA V23.3.2 -------GTTTGGTGTGTGGA--GACAGCATTGGACCAGATGAAAGGACAGAA---AACACATCTGAAGAAACAAAAACGGTCAAGTTGAGCTGCTCATATAGTGCA------AGTATTGAATATG----TTGTGCTTTACTGGTA--CAGACAGTATAA---ACATGGAGAACCTCAGTATTTACTTCG V23.3.8 -------GTTTGGTGTGTGGA--GACAGTATAGAGCCAAATAAAGGAATGGAA---AAAACAATTAAAGAAACAGAACCTGTTCAGCTGAGCTGTGCGTATACTACAGCCACAAGTAGAAGTTATG----TTTATCTTTACTGGTT--CAGACAATCTTT---CAATGGAGAGCCACAGTATTTGTTAAA V23.3.3 -------GTTTGGTGTTTGGG--GACAATATTGAGCCAGA---GGAGAAAGAT---GTTATGACAAAAGAAAGAGAAGCTGTCAAGCTGGCCTGCTCATACAGTACA------ACCAACAATAGAG----TTCGGCTTTATTGGTA--CAGACAGAATCC---AAATGCAGAACTTTTGCTTTTAACATA V22.0 -------GTGCAATGTGTCAA--GATCAGATTGGACCAAGCAGAACCAG---T---GTCACGAGTACAGAAGGAGATTCGGTCAATCTGGAGTGTTCATACGACTCA------AGCATCAACAATG----TCTTCCTATATTGGTA--CAGAAAATACTC---TAATGCACAGCCACAATATATACTTCT V23.0.1 ----------GTGGATATACT--GATAGGATTGGACCAGGGGAAGGAGACACAAATATAATCAGTATGGAAGGAGAGTCTGTGACACTGAGCTGCACATATGAAACA------AACAGTAATGACA----TCTGGCTTTATTGGTA--CAGACAGTTTTC---TAACAGAGAACCAGAATATATACTGTT V23.0.3 ----------GTGCACTTTCC--GACAGCATTGGACCAAATGAAAAAGAAACTAATGTGATGAGTAGAGAAGGAGAGTCTATGAGATTGAGCTGCTCATTTGAATCA------AGCAGCAATTATG----TTTTGCTTTACTGGTT--CAGACAGAACCC---TGATGGAGAGCTGCAATATTTACTATA V26.0 ----------GTGTGTTTGCT--GACAGCATTGCACCAAAAAAGGAGGGTAGAAATAAAACCAGAAAAGAAGGAGAGTCTGTGACACTGAGCTGCACATTTAACCGC------AGCGATACTGCGA----TTTCACTTTACTGGTA--CATGCAGTATCC---AAACAGAGAACTTCAGTATTTACTGTA V23.1.3 ----------GTGTGTTTGCT--GACAGCATTAAACCAAAAGGCAAAGATAAA---ATAATCAGAAGCGAAGGAGAGTCTGTGACACTGAGCTGCGAATATGAAACA------AGCAGCAATAATG----TAATGCTATACTGGTA--CAGACAATATAC---TAACAGAGAACCTCAGTTTTTGCTGCG V23.1.5 ----------GTGTGTTTTCT--GACAGCATTGGACCAAAAGATGAAGACAAACATATAATCAGGAGAGAAGGAGAGTCGGTGACATTGAGTTGCTCATATGAAACA------CAGAGCAATAATG----TTTGGCTTTACTGGTA--CAGACAATATAC---TAACAAAGAACCTCAGTATTTACTTTT V23.1.6 ----------GTGTATTTGCT--GACAGCATTCTTCCAGACAAAGATAGTAAT---ATGACGAGTAATGAAGGAGAGTCTGTGACACTGAAATGCTCTTATGATACG------AGCAGCAATAATG----TTTTGCTTTACTGGTA--CAGACAGTATGC---TAATACTGAACCTCAGTATTTACTGTG V23.1.10 ----------GTGTATTTGCT--GACAGCATTCGTCCAGACAAAGATACTA------TGACGAGTTATGAAGGAGAGTCTGTGACACTAAAATGCTCTTATGATACG------AGCAGCAATAATG----TTTTGCTTTACTTGTA--CAAACAGAATGT---TAATACTGAACCTCAATATTTACTGTG V23.1.7 ----------GTGTATTTGCT--GACAGCATTCGTCCAGACAAGGACACTAAT---ATAATCAGAAGTGAAGGAGAGTCTGTGACACTGAGATGCTCGTATGAAACA------AACAGCAACTATA----TTAGACTTTACTGGTA--CAGACAGTATGT---TAATACCGAACCTCAGTATTTACTGTG V23.1.8 ----------GTGTGTTTCCT--AACAACATTTGGCCAAAAGACAAAGAAGTG---ATAGTCAGTAATGAAGGAGAGTCTGTGACACTGACCTGCTCATACAGTTCA------GGCAGCAATGATG----TTTGGATTTACTGGTA--CAAATATCTTTC---TAACACAGAACCTAAGTTTTTACTGTG V23.1.1 ----------GTGTGTTTGCT--GACAGCCTTCAACCGAAGGACAAGGACAAA---ATAATCAG---CGAAGGACAGTCTGTGACACTGAGCTGCACATATGATACA------AGCAGCAGTTATG----TTTATCTCTATTGGTA--CAAACATAACAT---AAAGGGAGAACCTAAATTCGTACTGTG V23.1.4 ----------GTGTGTTTGCT--GACAGCATTGGACCAAATGACAAAGAAACTAGTGTGAGCAGTAATGAAGGAGAGTCTGTGACACTGAGCTGCTCATATGATGCA------CAGAGCAATAATA----TTAGACTATACTGGTA--CAGACAAAACAG---TAATCGAGAACCTGAGTATTTACTGTG V23.1.9 ----------GTGTGTTACCA--GACAGCATTGGACCAACTGACAAAGATGCAATTAAAACCAGGAAAGAAGGAGAATCTGTAACACTGAGTTGCTCATATGATACA------CAGAGCAATAATG----TTTATCTTTTCTGGTA--CAGACAATTTAC---TAACAGAGAACCTGAGTATTTACTAAT V23.1.2 ----------GTGTTTTGTCT--GACAGCATTCAACCAACAGACAAGAAAACTAATACTGACACAAAAGAAGAAGAGCATGTAACACTGAGCTGCTCATATGATACA------AGGAGCGAGTATG----TCTATCTCTACTGGTA--CAGACAATATCC---TAATAGAGAACCAGAATATTTACTGCG V23.0.2 ----------------GCACT--GATAGAATTGGACCAGAAGGAGAAACTAAT---ATACTCCAAGAAGAAGGACAGTCTGTGACACTGAGCTGCACATATGAAACA------AACAGCAACACCA----TTTATCTTTACTGGTA--CAGACAGTATCC---AAAGACAAAACCTGAATTTATACTGTA V16.0 ----------TTGCTTTTGGT--GATACCATTGGACCAGATGAGCCTTTA------ATTTTCAGAAGTGAAACTGACCGGGTGACACTGAGTTGTTCGTACCAAACA------AATAGTGAACATG----TTGATCTTTACTGGTA--CAGACACTATCC---TAAAAGTGAACCACAGTATTTACTATG V21.0 -------AAATCACTTTCTGT--GCTCATATTGCACCAAACACAAAATTC------GTCATTGGTAGAGAGGGGGAATCTGCAACTCTCTCATGCCTGTATGAATCC------AGCAGCTCCCATG----TGTTGCTCTTCTGGGG--CAGGATACTCCC---AAAACAGGCACCTGAGTTTTTACTGCT V30.1.1 ------GATGTGATTCTCAG---GACAAAGTTGATCAGGACACAAAGACTCAA---TCTGCTTTTGAAGGTGA---TACTGTAACCATTGACTGCACATATCAGACTTCA----AC---AAACCCA------ACTCTATTCTGGTA--CCAGCAGAAAGT---CAAAGGAGTCCCCAAACACATGCTAAA V30.1.3 ------GATGTGATTCTCAG---GATAAAGTTGATCAGCACACAAAGACTCAA---TCTGCTTTTGAAGGTGA---TACTGTAACCATTGACTGCACATATCAGACTTCTT-ATAC---AACACCG------ACTCTATTCTGGTA--CCAGCAGAAAGT---CAACGGAGTCCCCAAACACATGCTTAA V30.1.4 ------GATGTGATTCTCAG---GACAAAGTTGATCAGCACACAAAGACTCAA---TCTGCTTTTGAAGGTGA---TACTGTAACCATTGACTGCACATATCAGACTTCTT-CAAC---AGGCCCG------ACTCTATTCTGGTA--CCAGCAGAAAGT---CAATGGTGTCCCAAAATACATGCTGAA V30.1.2 ------GATGTGATTCTCAG---GACAAAGTTGATCAGCACACAAAGACTCAG---TCTGCTTTTGAAGGTGA---TACTGTAACCATTGACTGCACATATCAGACTTCTT-ATAC---AGGCCCA------ACTCTATTCTGGTA--CCAGCAGAAAGT---CAACGGAGTCCCCAAATACATGCTGAA V30.1.6 ------GATGTGATTCTCAG---GATAAAGTTGATCAGCACACAAAGACTCAA---TCTGCTTTTGAAGGTGA---TACTGTAACCACTGACTGCACATATCAGACTTCTT-CTAC---AGGCCCA------ACTCTATTCTGGTA--CCAGCAGAAAGT---CAGTGGAGTCCCCAAACACATGCTGAA V30.1.7 ------GATGTGATTCTCAG---GATAAAGTTGATCAGCACATAAAGACTCAA---TCTGCTTTTGAAGGTGA---TACTGTAACCATTGACTGCACATATCAGACTTCTT-ATAC---AACACCG------ACTCTATTCTGGTA--CCAGCAGAAAGT---CAATGAAGTCCCCAAACACATGCTGAA V30.1.5 ----AGGATGTGATTCTCAG---GACAAAGTTGATCAGCACACAAAAACTCAA---TCTGCTTTTGAAGGTGA---TACTGTAACCATTGACTGCACATATCAGACTTCTT-ATAC---AGGCCCG------ACTCTATTCTGGTA--CCAGCAGAAAAT---CAA----GTCCCCAGATATATGCTGAA V30.0.2 ------GCTGTGATTGTGCT---GAGAGTGTTGATCAGAATACAAGAGTTGAA---ACTTCTGTTGAAGAAGG---ATCTGTGAGCATTAGCTGTACATATCAAACCAGTG-ATCC---TTCCCCA------AATCTCTTTTGGTA--CCAACAAAAGCC---GAACACAATCCCCAAACACATGATGAC V30.0.3 ------GCTGTGATTGTGCT---GAGAGTGTTGATCAGAATACAAGAGTTGTA---ACTTTTGTTGAAGAAGG---ATCTGTGACCATTAACTGTACATATCAAACCAGCG-ATCC---TTCCCCA------TATCTGTTCTGGTA--CCAACAAAAGCC---GAACACAATCCCCAAACACATGATGAC V30.0.1 ------GCTGTGATTGTGCT---GAGAGTGTTGATCAGAATACAAGAGTTGAA---ACTGCTGTTGAAGGAGG---ATCGGTAACCATTAACTGTACATATCAAACCAGTG-ATCC---TTCCCCA------TATCTGTTCTGGTA--CCAACAAAAGCC---AAACACAATCCCTAAATACATGATGAT V35.0 ------GGTGTAAATGTAAG---GATTCTGTCCACCAGAA---GAGAGTTGAA---ACTGCTGTTGAAGGCAC---CGATTTATTCATCAGTTGCTCATATACAAGCACTG-ACCC---AATACCA------ACGCTTTTCTGGTA--CCAGCAAAAAGT---AAATGAGTTCCCTAAGTACATGCTAT- V30.0.4 ------GTCGTGTTTCTCTG---GATAAAGTTGAACAAAACTCAAGAGTTAAA---ACTGCTGTTGAAGGTGA---AGATGTAATAATCAACTGTCAGTATGAAACCACTG-ATAC---ATCGCCA------TATCTCTTCTGGTA--CCAGCAAAAAGT---CAACAGTGTCCCAGAATACATGCTGAA V39.0 ------GTCGTGTTTCTCTG---GATAAAGTTGAACAAAACTCAAGAGTTGAA---ACTGTTGTTGAAGATGA---AGATGTAATAATCAACTGTCAGTATGAAACCACTG-ATAC---ATTGCCA------TATCTCTTCTGGTA--CCAGCAAAAAGT---CAACAGATTCCCAGAATACATGCTGAA V30.0.5 ------GATTTGTTTCCCCG---GACAGTGTTGAACAGAAAACAAGAGTTCAA---ACTGCTGTAGAAGGTGA---AAGTGTAACAATCAACTGCACTTATTCAACCACTG-ATAC---ATTTCCA------TATCTCTTCTGGTA--CCAGCAACAAGT---AAACGGAATCCCTCAATATATGCTGAA V30.0.6 ------GAATAGAGTCGCAG---GACAATGTTGAGCAGAAAGCAAGAGTTCAA---ACTGCTGTTGAAGGTGA---AGCTGTTGAGATCGACTGCACATATCAAACC--CA-GCTC---ACCCCGA------C-TCTCTTCTGGTA--CCAGCAAAAGAC---AAACACAATCCCAAAATATATGCTCAA V29.0 ------AATGCAGAGCACAA---GATAGTGTTACTCAGACAGCTACAGTTCAA---ACTGCTACTGAAGGTCA---AGCAGTCACTATCGAATGCACATACAAAAC---CA-ACGC---ATTCCCA------ACGCTTTACTGGTA--CCAGTATAAAGC---AAATCAGTTCCCAAAGTATATGCTGAA V41.0 ------GATGCAAAGCACAG---GACAATGTAATACAGCCCAAACAAGTTATG---ACAGCTTATGAAGATGG---AACAATCACTCTTGAGTGCACGTACAAAGCGTCCA-GCACTCAATATCCA------AACCTCCTCTGGTA--CCAACAGATGAC---GAATGGATCCCCAAAATACATGCTGAT V32.0.2 ------GGTGCAGATCAGAA---GACAGAGTTGATCAGCCGGACAAACAACTG---ACTGAAGCTGAAGGAGG---ATCAGTGACATTACAGTGCAAATATAAAACAGATT-CAGT-GCAAGA--A------AATTTATTCTGGTA--CATCCAGAGAAC---AAATGACCTACCAAAATACATCCTGAG V32.0.4 ------GTTGCAGATCAGAG---GACAGAGTTGATCAGCCGGACAAACAACTG---ACTGAAGCTGAAGGAGG---ATCAGTGACATTACAGTGCAAATATAAAACAGATT-CAAC-ACAAGA--A------GATTTATTCTGGTA--CATCCAGAGAAT---AAATGACCAACCAAAATACATCCTGAG V32.0.60 ------AGTTCAGATCAGAG---GACAGAGTTGATCAACCGGAAAAGGAAATA---ACTGTAGCTGAAGGAGG---ATCAGTGACATTACAGTGCAAATATGAATCATATT-CTGT-GCAAAA--A------GATATATTCTGGTA--CGTGCAGAGAAC---AAATGACTTCCCAAAATACATCCTGAG V32.0.3 ------AGTGCAGATCAGAA---GAGAAAGTTGATCAGCCAGACAAACACATG---ACTGAAGTTGAAGGAAA---GTCAGTGACGTTACGATGTACATTTACAACAACTT-CAAA-CCAACC--A------GACCTCTTCTGGTA--CATCCAGAGAGC---AAATGATCATCCCAAATACATCCTGAG V32.0.5 ------AGTGCAGATCAGAA---GAGAAAGTTGATCAGCCAGACAAACACATG---ACTGAAGTTGAAGGAAA---GTCAGTGACGTTACGATGTACATTTACAACAACTT-CAAA-CCAACC--A------GACCTCTTCTGGTA--CATCCAGAAAGC---AAATGATCATCCCAAATACATCCTGAG V32.0.1 ------AATGCAGACCAGA------TAGTGTTAATCAGCCAGACACACACGTA---ACTAAATCTGAAGATGG---AGCAGTGACATTACAATGCAAATACACTGCAACTT-CCACAGCAACACCA------GAACTGTACTGGTA--CATCCAGAGAGA---ACATGAAATTCCCAAATACATCCTGAG V31.0 ------GGTGTCATTCAGAT---GACACGGTTGATCAGCCACTTAAACATGTT---ACCGCATTAGAGGGAAT---CTCAGTAACACTGGACTGCAAATATACCACATCCT-CAAC-ATCACA--A------GAGCTTTTCTGGTA--CATTCAGAGAGC---AGATGGATCTCTAAAA------CTTCA V33.0.6 ------TGTGCAGGGGGCAA---GACAAAGTTCAACAGCCTTCAGGAGAAAAG---ACTGAAAATGAAGGAGA---TCAAGTCAATTTAATGTGTAATTACA---CTACTA-CAGCTTCAGACACAAATATTTATCTCTTCTGGTA--TAAACAGCTGCC---AAACAGATCACCAACATTCATCCTGAA V33.0.7 ------TGTGCAGGGGGCAA---GACAAAGTTCAACAGCCTTCAGGAGAAATG---ACTGAAAATGAAGGAGA---TCAAGTCAGTTTAATGTGTAATTACA---CTACTA-CAGCTTCAGATGGAAACATTTTTCTCTTCTGGTA--CAAACAGCTGTC---AAACAGATCCCCAACATTCATCCTGAA V33.0.2 ------TGTGTAGGGCGCAA---GACAGAGTTGAACAGCCTACAAGAAAAAAG---ACTGAAAATGAAGGAGA---TCAAATCACTTTAATGTGTAATTACA---CTACTA-CTTCTACAAATGCAGATGTTTATCTCTTCTGGTA--CAAACAGCTGCC---AGACAGATCCCCAACATTCATCCTGAA V33.0.5 ------TGTGCAGAGGACAG---GACAGAGTTGAACAGCCTTTAAGAAGCATA---AGTGCAAATGAAAGAGG---TCAAGTTATAATAAGCTGTAGCTACA---GCACCA-CTGATATAAATATA------TACCTCTTCTGGTA--CAAACAGCTTCC---AAACAGATCACCAACATTCATTCTGAA V33.0.8 ------TGTGCAGAGGTCAA---GACAGAGTTGAACAGCCTTTAAGAGACATA---ACTGCAAATGAAGGAGA---TCAAGTTATTATACCCTGTAATTACA---GCACCA-CTGATACAAATCCA------TATCTCTTCTGGTA--CAAACAGCTGCC---AAACAGATCACCAACATTCATTCTGAA V33.0.9 ------AGTGCAGAGGACAA---GACAGAGTTGAACAGCCATCAATAGAAATG---ACTGAAAGTGAAGTGGA---AGAAGTTATTATAAGATGTAATTACA---GTACTC-TTTCTACAAGTCCA------GATCTCTTCTGGTA--CAAACAGCTGCC---AAACAGATCACCAACATACATTCTGAA V33.0.13 ------TGTGCAGAGGACAA---GACCGAGTTGAACAGCCTATAAGAGAAATG---ACAGAAAGTATAGGAGA---TAAAGTTGTTATCAGCTGTATTTATA---GCACCA-CTTATACAAGTCTG------ACTCTCTTCTGGTA--CAAAAAGCTGCC---AAACAGATCACCAACATACATTCTGAA V33.0.10 ------TGTGCAGAGGACAA---GACAGAGTGGAACAGGTTGAAGGAGAAATG---AGTGACAGTGAAGGAAA---TCATGTTATTATAAGCTGTAATTACA---GCAC----TTCACAAACTGCA------TATCTCTTCTGGTA--CAAACAGCTGCC---AAACAGATCACCAACATTCATTCTGAG V33.0.11 ------TGTGCAGAGGACAA---GACAGAGTGGAACAGGCTGAGGGAGAAATG---ACTGAAAGTGAACGAAA---TCATGTTATTATAAGCTGTAATTACA---TTACAA-CTTCAACAACAGTG------TATCTCTTCTGGTA--CAAACAGCTGCC---AAACAGATCACCAACATACATTCTGAG V33.0.12 ------TGTGCAGAGGACAA---GACAAAGTTGAACAGCCTATAAGAGAAATA---ACTGAAGGTGAAGGAGC---TCATGTTTTTGTCCAGTGTAATTATA----------CTTCATCAAATCCA------ACACTCTTCTGGTA--CAAACAGCTGCC---AAACAGATCACCAACATTAATTCTGAG V33.0.3 ------TGTGCCGGGGATCATCAGACAATGTTGAACAACCTTCAGGACAAATT---ACAGCAAATGAAGCAGA---TTCAGTTCATCTACAGTGCAGTTACT---CTACAA-CTTTCACAAACGCT------TATCTCTTCTGGTA--CAAACAACTGCC---AAACAGATCACCGACTTTCATCCTGAG V33.0.4 ------TGTGCAGGGGACAG---AACAGAGTTGAACAACCTTCAAGAGAAATG---ACGGCAGATAAAGGAGA---TACAGTTACTCTGCCGTGCAACTATT---CTGTCA-CGACCACAAATGCT------TATCTCTTCTGGTA--CAAACAACTTCC---AAACAGATCGCCAACATTCATGCTAAG V33.0.1 ------TATGTGGGGAACAA---GACAGTGTTGATCAGCCGGACAGAGAACTG---ACTTCTGCTGAAGGAAA---TGAAGTGATTTTATTGTGTAATTATAAACTGATGA-GCGCTGCTAATGCA------TATCTCTACTGGTA--CAAACAACTACC---AGACACATCACCAGCATTCATTCTGAG V18.0 ------AATGCAGATGTGAA---GACGCTGTACGTCAGCCAACAGCTTCTGTA---TCTGCTTTCACAGAAGA---AACCATAACCTTGAGCTGTGAATATTATACAACAG-ACTC--GATGCCTG-------CTCTGCTATGGTA--CCGGCAAACAAA---GAATTTCCCACCACAATATATTTTAGT V27.0 ------AATCCAGAGCACAA---GACTCAGTTTATCAGCCAAAAACCCTCCAA---TCAGCAAATGAAGAGGATGCAGCAGTAACCTTGGATTGTAAATACAGTACATCAA-GCTC--AAACCCTA-------CTCTACTCTGGTA--CAGACAAGATGA---TTATGGCTCTCCGAAATACATTCTACT

Sequences continued: V12.0.1 GGTTAGTGAAGCC------AAGCTGATCCAACCAGCTGATCCT----------CCAATTTCTGGACTATCTGCT-AAATTAAA--TGAGGAGAATAAC---CTTGTGTATCTGGAGATCTCCTCTGCTGCA-TATCAGACTCTG--CTGTGTATTA-CTGCGCTCTGAAGCC---------------- V12.0.2 GGTTAGTGAAGCA------AAGCTTGAACAACCAGCTGATCCT----------CCAATTCCTGGACTACCTGCT-AAATTAAA--TGAGGAGAATAAC---CTTGTGTATCTGGAGATCTCCTCTGCTGCACTATCGGACTCTG--CTGTGTATTA-CTGCGCTCTGACGCC---------------- V13.0 AGTTAACGAAGCT------GCATTTAAACAACCTGCTAATCCT----------CCCATTCCTGGAATATCTGCT-AGATTAAA--CGAGGAGAAAAAT---TGTGTGTACCTGGACATCACCAGCACTGCAATATCAGACTCTG--CGGTGTATTA-CTGTGCTCTGCAGCC---------------- V5.0.1 CATCACTGAATCT------GGAGAAGCAGTTAAAGCAGACCCA----------TCAGCCCACTGGCTATCAGCT-ACAGTCCA--CAAACAAAGCAAA---CTTACAGAACTGGAAATCAGCCCTGCTAAAGTGACTGACTCTG--CTGTGTATTA-CTGTGCCCTGAAGCC---------------- V5.0.2 CATCACTGAATCT------GGAGAAGCAGTTAAAGCAGACCCA----------TCATCCCACTGGCTATCAGCT-ACAGTCCA--CAAACAAAGCAAA---CTCACAGAACTGGAAATCAGCCCTGCTAAAGTGACTGACTCTG--CTGTGTATTA-CTGTGCCCTGAAGCC---------------- V17.0 AATCATGGAGTCA------GGACAAAATCAAACTGCGACACCG----------CCTCACCCCCGACTGACCACC-TCAGTTAA--TAAAGACAAAAAG---CTGGTGAATCTGGAAATCTTCCCCGCGGATGTGAAGGATTCTG--CTGTGTATTA-CTGTGCACTGCAGCC---------------- V3.0.1 TCACACTGAGAGC------AACAATCAGTCTTACTCCAG-------------------GCTTCGTCTGCTCGCT-GTGGCTGA--AAAAGAAATCAAA---CGTATGAATTTGAGCATATTTCATACAGAGATGCAGGATTCTG--CTCTTTACTA-CTGTGCCCTGGAGCC---------------- V3.0.3 TCACACTGAGAGC------AACAAGCAGTCTCACTCCAG-------------------GCTTCGTCTGCTCGCT-GTGACTGA--GAAAGGAATGAAA---CGTATGAATTTGAGCATATTTCATACAGAGATGGAGGATTCTG--CTCTTTACTA-CTGTGCCCTGGAGCC---------------- V3.0.2 TCACACTGAGAGC------AAAAATCAGTCAGACTTCAA-------------------GCTTCGCCTGCTTGCT-GTGGGTGA--GAAAGGAATGAAA---CGTATGAATTTGAGCATATTTCATACAGAGATGCAGGATTCTG--CTCTTTACTA-CTGTGCCCTGCAGCC---------------- V3.0.4 TTACACTGAGAGC------AACAATCAGTCAGATTCCAA-------------------GCTTCGTCTGCTCGCT-GTGGCTGA--TAAGGGACAGAAA---CTTATGAATTTAAGCATGTATTTTACAAAAGTGGAGGATTCTG--CTCTTTACTA-CTGTGCCCTGCAGCC---------------- V4.0.1 TTACTTTGAGAAC------AA------GCCACAAACTGA-------------------GGGCCGCATGACCGCT-GCATCTGA--TAAAGCAGTCAAA---CACATGAATCTGACCATCACTTCTACACAATTGACAGACTCTG--CTGTGTACTT-TTGTGCCCTGGTGCC---------------- V4.0.3 TTACTTTGAGAAC------AA------GCCACAAACTGA-------------------GGGCCGCATGACTGCT-GCATCTGA--TAAAGCAGTCAAA---CACATGAATCTGACCATCACTTCTACAAAAGTGAAAGACTCTG--CTGTGTACTA-CTGTGCCCTGCAGCC---------------- V4.0.2 TTACTTTGAGAAC------AT------GTTTACAACTAA-------------------GAACCGCATGATTGCT-GCGTCTGA--TAAAGCAGTCAAA---CACATGAATCTGACCATCAGTTCTGCAGAAATTACAGACTCTG--CTGTGTACTA-CTGTGCCCTGCAGCC---------------- V9.0.1 AACTTTTGAAA---------ATGCGGCACCAGAGCAGAA-------------------GGACCGTCTACTTGCT-AAAGCACA--AAAAGACAAAAAA---CAACTGGATCTGGAGATCTCCAAAGCTGAAATGTCAGACTCTG--CTGTGTATTA-CTGTGCACTGGTGCC---------------- V9.0.2 ACCTTTGAAA---------GTGCGGAACCAGCGCAACA--------------------GGGCCGACATCTTGCT-AAAGCACA--AAAGAACAAAAAA---CAACTGGATCTGCTCATCTCCAAAGCTGAAATGACAGACTCTG--CGATGTATTA-CTGTGCACTAGTGCC---------------- V8.0 AATACAGAGACT-------GGTGCTGGATCTGAACCCAC-------------------TTTACGTCTTACATCG-ATGGCCAA--AAAAGCACTCACA---CAAGTGGATCTGGAGATCTCCTCTACTGAAGTGAAAGACTCTG--CTATGTATTA-CTGTGCCCTGCAGCC---------------- V6.0.1 CATCTTTGAAAGT------GGAACTATGAGTAAAGACAAACC----------------AGACAGATTTTCTGCT-CAAGTTAA--TAAAGGCACCAAA---CAAGTGGATCTGAACATCTCCCCTGCTGTAGAAACAGATTCTG--CCATGTATTA-CTGCGCCCTGGTGCC---------------- V6.0.3 CATCTTTGAAAGC------GGAACTATGAGTAAAGACAAACC----------------AGACAGATTTTCTGCT-CAAGTTAA--TAAAGACAGCAAA---CAAGTGGATCTGAACATCTCCTCTGCTTTAGAAACAGATTCTG--CCATGTATTA-CTGTGCCCTGGTGCC---------------- V6.0.2 CATCTTTGAAAGC------GGAACTATGAGTAAAGACAAACC----------------AGACAGATTTTCTGCT-CAAGTTAA--TAAAGACACCAAA---CAAGTGGATCTGAACATCTCCTCTGCTGTAGAAACAGATTCTG--CCATGTATTA-CTGTGCCCTGGTGCC---------------- V6.0.4 CATCTTTGAAAGT------GGAACTTTGAGTGAAGACAAACC----------------AGACAGATTTTCTGCT-CAAGTTAA--TAAAGACACCAAA---CAAGTGGATCTGAACATCTCCTCTGCTGTAGAAACAGATTCTG--CCATGTATTA-CTGTGCCCTGGTGCC---------------- V7.0 TGCCACTG--AGC------TCAATGATCAATCAA-ACGCTGC----------------GCTTCGTATTTCAAAT-GTGGTCGA--TAAACA-AAAAAG---CA--TGAGTCTGAGCATTTTCCATGCAGAAATTCAAGATTCAG--CGACGTACTA-CTGTGCTCTGGAGAC---------------- V34.0.1 GCTCAATGAATAT------TCAAACAGCACTAAGTCTTA-------------------TCTTCGTCTATTTTCA-AACGCTGT--AAAAGCACTGAAG---CGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V34.0.3 TCTAAATGAATAT------TCAAACAGCTCTAAACCTGA-------------------TCTTCGTCTGGTTTCA-AACGCTGT--AAAAGCACTGAAG---CGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V34.0.4 TCTAAATGAATAT------TCAAACAGCTCTAAGCTTGA-------------------TCTTCGTCTGGTTTCA-AACGCTGT--AAAAGCACTGAAG---CGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V34.0.2 GCTCAATGAATAT------TATAACAAATCCAAGCTTGA-------------------TCTTCGTCTGTTTTCA-AACGCTGT--AAAAGCACTGAAG---CGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V34.0.5 TGTCACTGAATAT------TCAAACAGCTCTGAGTCTGA-------------------TCTTCGTCTGTTTTCA-AACGCTGT--AAAAGCACTGAAG---CGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V34.0.6 TGTCACTGAATAT------TCAAACAGCTCTGAGTCTGA-------------------TCTTCGTCTGTTTTCA-AACGCTGT--AAAAGCACTGAAG---CGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V10.0.2 GGTAATGGAGTCAGTCATCAGACATGTAACATATGCAGATCCT----------AAAATCCCTGGAATGGATGGA-GAAATGAG--CATGAGTGAAAAA---CAAGTGGATCTGGAGATCTTCTCTGCTACAATATCAGACTCTG--CAGTGTATTA-CTGTGCCCTGCAGCC---------------- V10.0.3 TGTAAGGGAATCAGCTAAAAAATCTGTAACATATGCAGATCCT----------AAAATCCCCGGAATGGATGGA-GAAATGAG--CATGAGTGAAAAA---CAAGTGGATCTGGAGATCTTCTCTGCCGCAGTATCAGACTCTG--CTGTGTATTA-TTGTGCCCTGGAGCC---------------- V10.0.1 TGTATTGAAGTCAGCTACAAAATCTGTAACATATGCAGATCCT----------AAAATCCCCGGAATGGATGGG-GAAATGAG--TATAAGTGAACAA---CAAGTAGATCTGGAGATCTCCTCTGCTGCAGTATCAGACTCTG--CAGTGTATTA-CTGTGCCCTGCAGCC---------------- V10.0.4 TGTAACAGAGTCAACCAGCAAAATTGTGACCTATGCAGATCCC----------AAAATTCCTGGAATGGATGGA-GAAATGAG--GATGAGTGAAAAA---CAAGTGGATCTGGAGATCTCCTCTGCTGCAGTATCAGACTCTG--CGGTGTATTA-TTGTGCCCTGCAGCC---------------- V10.0.5 TGTCATCGAGTCGCCAACAAAGACAGTTATACATGCAAAACCT----------CCCATTCCTAGAGTGGATGGA-GAAATGAG--TATGAGTGATAAA---CGTGTGAATCTGGAGATCTCCTCTGCTGAAGTGACTGACTCTG--CTCTGTACTA-CTGTGCTCTGGTGCC---------------- V38.0.1 -----CGCATACATTTATGAGCATGGAGCTACCAGTAAACCTC----------TTCCTCCACGACTTTTACCTA-AAGTTGA---TAAAAACAGCAAA---CGTGTGAGTCTGGAGATCTCTGAAGCTGAAGTGACTGACTCTG--CGCTGTATTA-CTGTGCACTGACGCC---------------- V38.0.2 -----TGCATACATTTATGAGCATGGAGCTACCAGTAAACCTC----------TTCCTCCACGACTTTTACCTA-AAGTTGA---TAAAAACAGCAAA---CGTGTGAGTCTGGAGATCTCTGAAGCTGAAGTGACTGACTCTG--CGCTGTATTA-CTGTGCACTGACGCC---------------- V38.0.3 -----CGCATACATTTATGAGCATGGAGCCACAAGTAAACCTC----------TTCCTCCTCGACTTTTACCAA-AAGTTGA---TAAAAACAGCAAA---CGTGTGAGTCTGGAGATCTCTGAAGCTGAAGTGACTGACTCTG--CGCTGTATTA-CTGTGCACTGACGCC---------------- V11.0.1 TGTTTATGAATCA------AACACAGTGTTTCATGCTGAACCT----------CCTTTCCCAAGACTGAACGCC-AGTGTGAA--TAAAGTGGAGAAG---CAGGTGAATCTGACCATCTCTTCTGCTGCAGTCACAGATTCAG--CTGTTTATTA-CTGTGCTCTGCAGCC---------------- V11.0.2 TGTTTATGAATCA------AACACAGTGTTTCATGCTGAACCT----------CCTTTCCCAAGACTGGACGCC-AGTGTGAA--TAAAGTGGAGAAG---CAGGTGAATCTGACCATCTCTTCTGCTGCAGTCACAGATTCAG--CTGTTTATTA-CTGTGCTCTGCAGCC---------------- V11.0.3 TGTTTATGAATCA------AACACCGTGTTTCATGCTGAACCT----------CCTTTCCCAAGACTGAACGCC-AGTGTGAA--TAAAGTGGAGAAG---CAGGTGAATCTGACCATCTCTTCTGCTGCAGTCACAGATTCAG--CTCTGTATTA-CTGTGCTCTGCAGCC---------------- V14.0 CATCTACGAAAGC------GGACTGAAGAGTGACAACATACC----------------AGAGCGCTTAACGCCG-AAAATCAA--CAAAACTAAGAAA---GTTGTGGATCTGGAGATCTCCTCTGCTACAGTATCAGACTCTG--CTGTGTATTA-CTGTGCCCTGAAGCC---------------- V36.2.8 CACCTACAGAACAGCAACAAAAGCTG---CACAGTCCGAT-------ATAGA------CACAAGATTCACTACT-AATGTTAC--AAAAAAGGAG------CATGTGGATCTGCTGCTCTCCTCTGCTGCAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGGC---------------- V36.2.10 CACCTACAGCTCTGCAACAAAAGCTG---CTCAGTCCGAT-------ATAGA------CACAAGATTCACTACT-AATATTAC--AAAAAAGGAG------CATGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V36.2.2 CATCTATGGGTCTGCAACAAAAGCTG---CACAGTCTGAT-------ATAGA------TACAAGATTCACTGCT-AATATTAC--TAAAAAGGAG------CATGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V36.2.11 CACCTACAGCAGTGCAAAAAAATCTA---AGGAGTCTGAT-------ATAGA------TCCAAAATTCAGTGCT-AATGTCTC--AAAAAGTGAA------CATGTGGATCTGCTGCTCTCTTCTGCTGCAGTATCAGACTCTGGTCCGTGTATCATCCGTTCATTTGATTATTCCTTTTATTACGGA V36.2.9 CACCTACAGAACAGCAAAAGAAGCTG---AGAAGTCTAAA-------GTAGA------TCCAAGATTTATTGCC-AATGTCCC--AAAAAGTGAG------CATGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTATATTA-CTGCGCTCTGAGGCC---------------- V36.2.1 AATCATTAGTAATTCAAATAAAGCAC---AGCAATCTCTA-------GTAGA------TCCAAGATTCACTGCT-TACATTGA--AAACAGAGACAAAATTCATGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V36.2.12 CATCTACAGTAGTGCAAAAGCTGCCG---AGAAATCTAAA-------GTAGA------TCCAAGATTCTTTGTT-AAAGTTGA--AAAAAGACAACAAATCCATGTGGATCTTCTGCTCTCCTCTACTGCAGTATCAGACTCTG--CTGTGTATTA-CTGCGCTCTGCAGCC---------------- V36.2.6 TACCTATGGCAGTGCAAAAGACACTG---AAACATCAAAA-------GTAGA------TCCAAGATTCTCAGTT-AAAGTTGA--AAAAAAGGAACGAAACCATGCAGATCTGAAGATCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGCGCTCTGAGGCC---------------- V36.2.7 CACTTTCGGCAGTGTAAAAGACGCTG---AAACATCAAAA-------GTAGA------CCCAAGATTCTCTGTT-AAAATTGA--AAAAAAGGAACAAAACCATGCAGATCTGAAGATCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGCGCTCTGAGGCC---------------- V36.2.5 AATTTTTGGCAGTACGAAAGACACTG---AAACATCAAAA-------GTAGA------TCCAAGATTCTCTGTT-ACAGTTGA--GAAAAAGGAACGAAACCATGCAGATCTGCTGCTCTCCTCTGCCGCAGTATCAGACTCTG--CTGTGTATTA-CTGCGCTCTGCAGCC---------------- V36.2.4 CATCTATGGGACTGCAAACAAAGGTG---AAACATCAAAA-------GTAGA------TCCAAGATTCTCTGTT-AAAGTTGA--AAAAAAGGAACGAAACCATGCAGATCTGAAGATCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V36.2.3 CATCTTTGAGGGTGAAAGAAATAGCA---AATCATCTGAT-------GTAGA------TCCAAGACTCTCTGCT-AAACTCAC--TAAAAGTGAGCATAACCATGCAGATTTGATGCTCTCCTGTGCTGCAGTATCAGACTCCG--CTGTGTATTA-CTGCGCTCTGAGGCC---------------- V25.0.2 CATTCTAGAATCCAATGGAGATGTTTTACAAAAATCTGAA----GCTGTGAA---AGATCCTCGATTCTCTGCA-AAAGTGAA--TGAAAAGAAAACC---CATGTGTTTCTGGAGATCTCCTCTGCTAAAGTCACAGATTCAG--CCATTTACTA-CTGTGCCTTGGAGCC---------------- V25.0.3 CATTCTCCATTCCAATGGAGAAGTTTTACAAAAATCTGAA----GCTGTGAA---AGATCCTCGATTCTCTGCA-AAAGTGAA--TGAAAAGAAAACC---CATGTGTTTCTGGAGATCTCCTCTGCTAAAGTCACAGATTCAG--CAATTTACTA-CTGTGCCTTGGAGCC---------------- V25.0.1 CATTCTACATGCTACAGGGAAAGTTTTACAGAAATCTGAT----ATTGTGGATCAAGATCCTCGATTCTCTGGA-AAACTGAA--TGAGGAGAAAACC---CACACGAGCCTTAATATCTCCTCTGCTAAAGTGACAGACTCAG--CAATGTATTT-CTGTGCCATGGAGCC---------------- V25.0.4 AAT---ACATAGGACTGGAAGTGTTTCCCGAAGTTCTACA----ATTGTGAATGAAGATCCTTGGTACTATGGA-AAACTGAA--TAAAGAGAAAACC---CATGTGTTTCTGGAGATCTCCACTGCTAAAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V15.0 GATACTA------ACAAAAAGTG------AAACCTCTGAA----AACATGCA------GCCTGGAATGTCAGTG-AGATTACA--TAAAGAGAAAGCC---TCTATGGATCTGAAGATCTCCTCTGCCAAACTAACAGACTCGG--CAGTTTACTA-CTGTGCGTTGAAGCC---------------- V19.0 AGTTCTGCAAACCACTGGACGGGTC---CAGACTGCACAG----AATCTGGA------TTCTCGGTTTGAGGCA-ATGCTGAA--TGAAAAGAAAAAC---AAAGTTCATCTGATCATCTCCTCTGTCAAAGTGACAGATTCAG--CACTGTATTA-CTGTGCTATGGAGCC---------------- V20.0 CATCCAACAAAGCACTGGACAAATGATACAGCGTTCAGAC----GACA---------------GGCTGTCAGGA-AAACTTAA--CAAAGAGAAAAAC---CGTGTAGATCTGCAAATCTCCTCTTCCACCATAAAAGACTCTG--CGCTGTATTT-CTGTGCTCTGCAGCC---------------- V37.0 CATATCAGAGGGTCTAACAGAGACTA---AGATTTCTGAAGTAGATTCCAGGTTTACAACTAAGCTGAGAAAAG-AAAATCAAGCTGGAAAAGAAATCAAGCGTGTGGATCTGCTGCTCTCCTCTGCTGCAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V36.1.2 CATTCCAGACAGTTCTAAAGAAGATT---GGAAGTCTAAT-------GTTGA------TGACAGATTTACAGCC-AGAGTCAC--TAAAAACAAAGAAAAGCGTGTGGATCTGCTGCTCTCCTCTGCTGCTGTATCGGACTCTG--CTGTGTATTA-CTGTGCTCTGGAGCC---------------- V36.1.3 TATTCCAGAGAGTAGAAAAGATGATA---CAAAGTCTGAT-------GTAGA------TCACAGATTCATAGCC-AGAGTCAC--TAAAGATAAAGAAAAGCGTGTGGATCTGCTGCTCTCCTCTGCTGCTGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGGAGCC---------------- V36.1.1 CATTCCAGAAGGTACTAAAGATGATT---GGAAGTCAGAT-------GTTGA------TAAAAGATTTACAGCC-AGAATTAC--TAAAGACAAAGAGAAGCATGTGGATCTGCTGCTCTCCTCTGCTGCTGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V36.1.6 CTTTATTCATGGTACAAGTGAAGCTA---AAGAGTCTGGT-------GTAGA------TCCCAGATTCACTGCT-AAACCCAA--TGGAGAAAAACAAAGCCATGTGGATCTGCTCCTCTCCTCTGCTGCTGTATCAGACTCTG--CTCTTTACTA-CTGTGCCATGGAGCC---------------- V36.1.4 TACTGTAGATACTGTAAACGAAGCTC---GAACGTCTGAT-------GTAGA------TGTCAGATTTGCTACT-AAAACCAG--AAAAGAAAATGCAAACCATGTGGATCTGGAGATCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAAGC---------------- V36.1.5 CATTGCAGAAAGGTCTAAAAAGGTAG---AGAGGTCGAAT-------ACTGA------TCACCGATTTACTACA-AAGATCAG--GATAGAGAAAGAAAAGCATGTGGATCTGCTCTTGTCCCCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGACGTCCATGAC---------- V24.0.1 GGATTATTATGGGTCAATTACTCATGC-GAATCCACCTAT------------------TGCTGGAATCTCCATT-AAACACAG--AAAAGACAACAGC---AGTGTGGATCTGGAGATCTCCTCTGTTGCAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC----------------V24.0.2 GGATTACTCTGGATTTGTTACTCATGC-TAATCCTCCATT------------------TGATGGAATCTCCATT-AAACACAG--AAAAGAAAACAGC---AGTGTGGATCTGCTGATCTCCTCTGCTGCAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V24.0.4 GGATTACTATGGGGCAGTTACTCATGC-AAAACCTCCGAT------------------TGCTGGAATCTCCATC-AAACACAG--AAAAGACGACAGC---AGTGTGGATCTGGAGATCTCCTCTGCTGCAGTATCAGACTCTG--CTCTTTACTA-CTGCGCACTGGAGAC---------------- V24.0.3 GGAGTATTCAGGAGTTATTACTCCAGC-GGATCCACCAGT------------------TCCAGGAATCTCCATC-AATCACAG--AAAAAAGCAAAGC---CATGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V24.0.5 AGAAGATTCAGGAGTAACAACTCCAGC-AAATCCACCAGT------------------TCCTGGAATCTCCATC-AATCACAG--AAAAGCAGAAAAA---CATGTGGATCTGCTGCTCTCCTCTGCTGCAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V28.0 GGAATCGTCTGGAGTTATAATACATGC-TACTCCTCAGGT------------------TCCAGGGATAAAAATT-ATCAATAA--AAAAAACACCAAG---CATGTGGAGCTGGAGATCTTCAGTGCATCAGTGGAACATTCAG--CTGTGTATTA-TTGCGCTGCGCGGCC---------------- V2.0.1 GATTGTGAGGTCTACTAAAACTACAATTGAAGCTTCTCCA-------CCACA------GCCACATATGTCCATC-AACCTCAG--TG---AGAAA------CTTGTGGATCTGGAGATCTCCTCTGTTGCAGTGTCAGACTCGG--CTGTGTACTA-CTGTGCCCTGGAGCC---------------- V2.0.3 GGTTGTGAGGTCTACTAAAACTAAAGTAGCAGCTTCTCCA-------CCTCA------GCCACATATGTCCATC-AACCTCAG--TG---AGAAA------CGTGTGGATCTGGAGATCTCCTCTGCTGCAGTGTCAGACTCGG--CTGTGTACTA-CTGTGCCCTGGAGCC---------------- V2.0.4 GATTGATAAATCTACCAAAGTTGTAGTGACCGCTTCTCCT-------CCGCA------TCCGCACGTGTCTATT-AATCTCCA--TG---ATAAC------CGTGTGGATTTGGAGATCACTCCTGCTGCAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V2.0.2 GATAGATGAATCTTCAAAGTACGTCACCAAAGCTGAACAA-------CCTCA------CCCACATATGTCCATCCAACTTCAGAACA---ACAAA------CGTGTAAATCTGATCCTCTCATTCGCTGCTGTATCAGATTCTG--CCGTTTACTA-CTGTGCCCTGCAGCC---------------- V1.0 GATTGATGAAGCTTCTGAATACGTGACAAATGCCATTCCA-------CCTCA------TCCACGCCTGTCAGTCCAACTTCACAAAGCACAAAAA------CGTGTTAGTCTAGAGATGTCGTCTGCAGCCGTTACGGATTCTG--CTCTTTACTT-TTGTGCTTTGAGGCC---------------- V23.2.6 GAAAGAGGCT--CGATCATGGAGTG-CCACTGGGACACCTGAT-------------GATCCTCGCATTCGGGCT-TCAACAT--------CAGAAACC---ACCACTGAACTCATTATTACTGGTGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTGATAGTTGGAG-----------

V23.2.4 GAAAGATGCT--CGATCATGGAGTG-GTGTGGCGACTCCTGAT-------------GATCCTCGCATTCGGTCT-ACAACAT--------CAGAAACC---ACCACTGAACTCATTATTACTGGTGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTGAGAGTTGGAG----------- V23.2.8 GAAAGATTGT--AGATCATATAGTA-ACACTGTGACCTCCGCT-------------GATCCTCGCGTTGAGCCC-AAAACAT--------CAGAAACC---ACTGCTGAACTCATTATTCAGAGTGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTACGA------------------ V23.2.7 GAAAGAGGCT--CGATCATTGAGGG-GTGCTGGAAACTCTTTT-------------GACAGCCGGTTTCAGTCG-ACCACAT--------CTGAAAGC---TCCACTGAACTCACTGTTACTGGTGTATCTCTGTCAGTTTCAG--CTCTTTATTA-CTGTGCTCTATTTGTTGGAG----------- V23.2.1 TAAACCTGCA--AAATCAGCTTCTG-TAAGTGGAGATCCAGTT-------------GATCGTAGGTTTCAGTCG-AGCACAT--------CAGACTCA---TCCACTGAACTCACTATTAGCGGTGCAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTTGGAG----------- V23.2.2 CAAAGCTGCA--CGATCAAGTAGTG-GAGGTGGGAGACCCGAT-------------AATCCTCGTTTTAAGTCG-ACTACAT--------CAGACTCA---TCCACTGAACTCACTATTAGCGGTGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGGAG----------- V23.2.3 T---TCTGCA--CAATCAAGTAGTG-GAAA----------------------------------ATTTCAAACA-GCTAAAT--------CACGCTCA---TCTACTGAACTCACTATTAGCGGTGTAACTCTGTCAGATTCAG--CTCTTTATTA-TTGTGCTCTAAGAGTTTTAG----------- V23.2.5 T---TCTGCA--GGTTCAAATATTG-GAAG----------------------------------GTTTCAAATC-GCTAAAT--------CGGACTCA---TCTACTGAACTCACTATTAGCGGTGTAACTCTGTCAGATTCAG--CTCTTTATTA-TTGTGCTCTACGAGTTGGTG----------- V40.0 TAAAGGAGCT--CGATCAAACTCAG-CCAAAGACACCTCTGAT-------------G---CCCGATTTGAGTCA-AGTACAT--------CTCGCATA---TCCACTGACCTCATTATTAAGACTGCAGCTCTGTCAGATTCAG--CTCTTTATTA-TTGTGCTCTAAGAGTTGAAG----------- V23.3.4 CAAAGGTGCA--CGATCAT---ATA-GTACTCATCACAAGTCT-------------GATGATCGGTTTGAGTCG-GCTACAT--------CACGTTCA---TCCACTGAACTCACTATTACTAATTTGCGTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAAGAG----------- V23.3.7 CAAAGGTGCA--CAATCAT---ATC-TTACTCATCACACTTCT-------------GATAAACGGTTTGAGTCG-GCTACAT--------CACGTTCA---TCCACTCAACTCACTATTAATGATGTGAGTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGCGTAGGAT----------- V23.3.6 CGAAAGTGCA--CGATCGG---CTT-CTGATTCACACTCCTCT-------------GATGATCGGTTTCACTCG-GCTACAT--------CACGTTCA---TCCACTGAACTTACTATTAATAATGTGCGTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGGAG----------- V23.3.5 CAGATATGCT--CGATCATTGGGTA-ATAATGAATACAAGTCT-------------GATGATCGGTTTCACTCG-GCTACAT--------CAAGTTCA---TCCACTGAACTCACTATTACTAATGTGCGTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAATAG----------- V23.3.1 CAGATATGCT--CGATCATGGGGTA-ATGATGAATACAAGTCT-------------GATGATCGGTTTCAGTCT-GCAACAT--------CAGAGTCA---TCCACTGAACTCACTATTACTAATGTGCATCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAAGAG----------- V23.3.2 CAGATATGCT--CGATCATATAATG-ATTATGAAGCCACTTCT-------------GATGATCGCTTTCAATCA-GATTCAT--------CATATACA---TCCACTGAACTCACTATTAAAGATGCACGTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAATAGTAGGAT----------- V23.3.8 CAAAAATGCA--CAAACAG---CCA-GTGATTCACACACGTCG-------------GATGATCGGTTTCAGACG-ACTATAA--------CACAGACA---GCCACTGAACTCACTATTACTGATGTACGACTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGGAG----------- V23.3.3 CAAAGGTGCT--CGATCTC---TGA-GTGCTAAACACTCCTCT-------------AATGATCGGTTTCAATCC-ACAACAT--------CAGACTCA---TCCACTGAACTCACTATTACTGATGTGCGTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGGAG----------- V22.0 GAAAGGCGGC--CGATCACAGACAT-ATGAGGACATACCAGAC-------------CGGGCTAAATTTGGATCA-GCTACGT--------CGCAGAAA---TCTACTACACTGACCATTAAAAGTGTGACTGTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGGAG----------- V23.0.1 TAAAGGTGCT--CGATCAAGGAGC------GAGGAGGATT-TA-------------CCTGACGGTTTTCAGTCA-AGGACAT--------CAGACTCA---ACCACTGAAATCACTATTAACAGTGTGCGTCTGTCAGATTCAG--CTCTCTATTA-CTGCGCTCTTGA-AGTAG------------- V23.0.3 TAAAGGTGCT--CGATCGTGGAGCTCTAAAGAAGATCATT-CA-------------GAAGATCGTTTTCAGTCA-ATTGCAG--------CATATTCC---TCCACTGAACTCAATACTGCTGAAGCGGCTCTGTCAGATTCAG--CTCTCTATTA-CTGTGCTGTGAAGAGCCGTCTGTACAG---- V26.0 TAAAGGTCAT--CGATCGTCTGAG----GAGGGGATTGTAACA-------------GACAGTCGTTATAAATCA-ACTGTCT--------CACCTACG---TCCACTTCACTCACCATTAACTCTGTGAATCTGTCAGATTCAG--CCCTGTATTA-CTGTGCTCTTAAAAAAGGAC----------- V23.1.3 TAAAGGAGCT--CGATCAAGGAATG-GACTTGATGACATACCA-------------GACCGTCAGTTTGATTCG-ACTTCAT--------CCCAAACC---TCCACTGAACTCACTAATACTGGTGTAAGCCTGTCGGATTCAG--CTCTCTATTA-TTGTGCAATAAGAGTTGGAG----------- V23.1.5 GAAAGGAGCT--CGATCAAATAGCA-GATCTGAGGACAGACCA-------------GATCATCACTTTCAATCA-TCTACAG--------GCCAAACC---ACCACAGAACTAACAATTGATAAAGCAGCTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTTGGAG----------- V23.1.6 GAAAGGAGCT--CGATCTAACAGCA-ACTATAAGAAAATACCA-------------GGCAGTCAGTTCGATTCC-ATTACAT--------CCCAGACG---TCCACTGAACTCATTATTAAGAGTGTATCTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGGAG----------- V23.1.10 GAAAAAAGCT--CGATCATACAATA-GAGATGAAGACATACCT-------------GACAGTCGGTTTGATTCG-ATTACAT--------CCCAAACC---TCTACTGAACTCATTATAAAGAGTGTATCTCTGTCAGATTCAG--CTCTCTATCA-TTGTGCTCTAACAGTAGGAG----------- V23.1.7 GAAAGGAGCT--CGATCAGACAGCG-ACTATAAGAATATACCA-------------GACAGTCGATTTGATTCA-ATTACAT--------CCCAAACC---TCCACTGAACTCATTATTAAGAGTGTATCTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAAGAG----------- V23.1.8 GAAAGGTGCT--CGAGCACAGAACA-TGCA---GCATATCCCA-------------GATCAGCGCTTTGAATCT-ACTACAT--------ACCAAACA---TCCACTAACCTCTTTATTAAGAGTGTATCTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTAGTAG----------- V23.1.1 TAAAGGTGCC--CGATCCCGAAGTG-ATAGACAGTATATACCA-------------AACCGTCAGTTTGATTCA-ATTACAT--------CCCAAACC---TCCACTGACCTCATTATTAAAAGTGTAACACTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTATTAGTTGAAG----------- V23.1.4 GAAAGGAGCT--CGATCATATGGAG-GAGA---GGATAAAACA-------------GACTCTCGGTTTAATTCG-AAAACAT--------CCCGAACT---GCAACTGAACTCACTATTTCTGCAGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTTTTAG----------- V23.1.9 TAAAGGAGCT--CGATCATATGGAG-GAGA---GGATAAAATA-------------GATTCACGGTTTCGTCCG-ACTACCT--------CCCAAACC---TCCACTGAACTCACTATTCCTGGTGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTTGTAG----------- V23.1.2 TAAAGGTGCC--CGTTCGTGGAGCA-GTTACAATAATATGCCA-------------GATCCTCAGTTTCAGGCG-ACTTCAT--------CCCAAACC---TCCACTGAACTCACTATTACTGCTGTAACTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCTCTAAGAGTTGAAG----------- V23.0.2 TAAAGGTGCT--CGATCATGGAGCA----GTGAGGATATTCCA-------------GATAGTTA---TGAATCA-GCTACAT--------CACATACA---TCCACTAAACTCATTATTAAGAGTGTATCTCTGTCAGATTCAG--CTCTCTATTA-TTGTGCCCTATTAGTTAGAG----------- V16.0 GAAGGGAGCC--AGAGCAAGGGATG-GTGAGAAGCATATGCCA-------------GATTCTCATTTTGAATCT-TCTACAA--------GCAGAACG---TCCACTGAGCTGATTATTAAAGTAGCGGCTCTGACAGATTCAG--CTCTCTATTA-TTGTGCTCTTTGGAAAAG------------- V21.0 CAAGGGTGCA--CGATCCGAGACAC----AATTTCAAAGCGTA-------------GATCCTCGATACGATTCT-GCCACTT--------CAAAGACC---GCCACTTTTCTGATCATTAACAATCTAACACTGGAGGACAAGG--CTTCCTACTA-CTGTGTGCTGGAAGAA--------------- V30.1.1 TAAAATCACCAACACAACTGGACACATTGAGGAAGAATTCAAT-------------GAG---AGATTTCACGCA-ACTCTCAG--CAAAACT---------TCAGTTCCTCTGACAATCAAGGATCTACGAGTGTCAGACT--G--CTATGTATTA-CTGTGCTCTGAGGCC---------------- V30.1.3 TAAAATCTCT---ACATCTGGACACACTGAGGAAGAATTTAAA-------------GAG---AGATTTCACGCA-ACTCTCAG--CAAAACT---------TCAGTTCCTCTGACAATCAAGGATCTACGAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V30.1.4 TAAAATCACT---ACGACTGGACACACTGAGGAAGAATTCAAG-------------AAA---AGATTTAACGCA-ACTCTCAG--CAAAACT---------TCAGTTCCTCTGACAATCAAGGATCTGCGTGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V30.1.2 TAAAATCACT---ACAACTGGACACACTGAGGAAGAATTCAAA-------------GAG---AGATTTCACGCA-ACTCTAAG--CAAAACT---------TCAGTTCCTCTGACAATCCAGGATGTGCGAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V30.1.6 TAAATTCTCT---ACAACTGGAAATAGTGAGGAAGAATTAAAA-------------AAAAAAAGATTTCATGCA-ACTCTCAG--CAAAACT---------TCAGTTCCTCTGACAATTCAGAATCTGCGAGTTCAAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAACC---------------- V30.1.7 TAAATTCTCT---ACAACTGGAAATAGTGAGGAAGAATTCAAG-------------AAA---AGATTTAATGCA-GCTCTCAG--CAAAACA---------TCAGTTCCTCTGACAATCAAGGATCTGCATGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAGCC---------------- V30.1.5 TAAAATCACTAACACATCAGGACACACTGAGGATGAATTAAAG-------------GAG---AGATTTCATGCA-GCTCTCAG--CAAAACT---------GACATGACTTTTATACTTCATTACACTTAAGAAACAAACT-TG--CCACAAAATT-ATTCCTACAGAATCC---------------- V30.0.2 GATAATCACT---ACAACTGTACAAAATGACAAAGACTTTGAG-------------GAGGAGAGATTTGGTGCA-AAACACGA--CAAAACTTTAAAA---TCAGTTCCTCTGCTGATCCAGGATCTGCGTGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V30.0.3 AATAATCACT---ACAACTGTACAAAATGACAAAGACTTTGAG-------------GAGGAGAGATTTGGTGCA-AAACACGA--CAAAACTTTAAAA---TCAGTTCCTCTGCTGATCCAGGATCTGCGTGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V30.0.1 GATATTTGCT---ACAACTGTACAAAATGACAAAGACTTTGAG-------------GAGGAGAGATTTAGTGCA-AAACACGA--CAAAACTTTAAAA---TCAGTTCCTCTGCTGATCCAGGATCTGCGTGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V35.0 --------CT---ACTTTTGAAAGCAAAGACGACGAATTCAAA-------------AA---GAGATTTAATGCA-CGACTGAA--CACATCTTCAACT---TCAGCTCCTCTGCTGATCCAGGATCTGCGAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAGCC---------------- V30.0.4 CAGATTC------AGTTCTGATGAGG------AGCAATTCAAG-------------GG---TAGATTTAGCAGC-GAACTCAA--CACAAGTTCAAAA---TCATTTCCACTGAAGATCCAGGATGTGCGTGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V39.0 CAGATTT------AGTTCTGATGAAG------AGCAATTCAAG-------------GA---TAGATTTAGCAGC-GAACTCAA--CACAAGTTCAAAA---TCATTTCCACTGAAGATCCAGGATGTGCGTGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V30.0.5 GAAATTTCCT---GGATCCGGTGAGATTGAGAATCAATTTAAA-------------AA---GAGATTTGATGCC-GATCTCGA--CTCCTCTACAACA---TCAGTCCTGCTGAGGATCAAAGAGGTGCATGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAGCC---------------- V30.0.6 CAGAGTTGGA---AAGACTGGAGGTGAAGACAAAGAATTCAAA-------------GA---AAGATTTACTGCA-AATCTCAA--CACATTTTCAAAA---TCAGTTCCTCTGATGATCAGAGATCTACATGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V29.0 GAGATAT---GCTGGATCAAGCGACGAAGATGACAATTTTAAG-------------GAC---AGATTTAATGCA-AATCTCAA--CAGCTCTTCTACA---TCAGTTCCTCTGACAATAACAGATGTGCGAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V41.0 GAGACTT---TCTGGATCTAGTGACACTAATGAAGAGTTTAAG-------------CAG---AGATTTAACGCA-GAACTCAA--CACATCTGCATTA---TCAATTCCTCTGACGATCAAGGATCTGCGTGCATCAGATTCTG--CTGTGTATTA-CTGTGCCCTGAAGCC---------------- V32.0.2 GAGAAGCAAATACGGAG---GAGAAAATGGCACAGAGTTTCAG-------------GAG---AGATTTCACTCT-GAACTTT--------CAACTAAT---TCAGTTCCTCTGACGATCAGAGATCTGCATGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAAGCC---------------- V32.0.4 GAGAAGCAAATATGGAG---GAGAAAATGGCACAGAGTTTCAG-------------GAG---AGATTTTACTCT-GAACTTT--------CAACTAAT---TTTGTTCCTCTGACGATCAGAGATCTGCATGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V32.0.6 GAGAAGTAACTTTGGAG---GAGAAAATGGCACAGAGTTTCAG-------------GAG---AGATTTCACTCT-GAACTGT--------CTTTAGAT---TCAGTTCCTCTGCTGATCAGAGATCTGCGTGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTTTGAGGCC---------------- V32.0.3 GAGGGACAAGTATGGAGCTGGAGAAAATGGCACCGAATTTCAG-------------GAG---AGATTTCACTCT-GAATTTT--------TAACAGAT---TCAGTTCCTCTGCTGATCAGAGATCTGCATGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V32.0.5 GAGGGACAAGTATGGAGCTGGAGAAAATGGCACAGAGTTTCAG-------------GAG---AGATTTCACTCT-GAACTTT--------TAACAGAT---TCAGTTCCTCTGCTGATCAGAGATCTGCATGTGTCTGATTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V32.0.1 GAAAAACAAATATGAAG---GAGAAAATGGCACAGAGTTTCAC-------------AAG---AGATTTCATGTT-GAATTAA--------CAACGGAT---TCAGTTCCTCTGCTGATCAAAGATCTGCGTGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAGCC---------------- V31.0 AAGAAATTCATACGGCGGAGGAGTGAATGGAACAGAGTTCAAG-------------GAA---AGATTTTACTCT-GAGGTGAA--AA---CATCAAAA---TCAGTTCCTCTGCTGATTCAGGATACGTGTGTGTGTGACTCTG--CTGTTTACTA-CTGCGCTCTGAGGCC---------------- V33.0.6 TAAATCTCCATTTAGTGAAGGAACTATTGAGCCTGAATTTAAC-------------AAG---AGACTTTCAGCA-ACACTGAA--TTCAACATCAAAA---ACAGTTCGTCTGCTGATCCAGGATCTGCGAGTGTCAGACTCGG--CTGTGTTTTA-CTGTGCTCTGAGGCC---------------- V33.0.7 TAAAATTCCATTTAGTGAAGGAACTATTGAGCCTGAGTTTAAC-------------AAG---AGATTTACAGCA-AAACTGAA--TTCAACATTAAGA---ACAGTTCCTCTGCTGATCCAGGATCTGCGAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTTTGAAGCC---------------- V33.0.2 TAAATTTCCATTTAGTGAAGGAACTATTGAGCCTGAGTTTAAC-------------AAG---AGATTTTCAGCA-AAACTGAA--TTCAACATTAAGA---ACAGTTCCTCTGCTGATCCAGGATCTGCGAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V33.0.5 TAAACAAACATATGGTGAAGGAATTACTGCGGCTGATTTTAAG-------------GAG---AGATTTTCTGCA-ACACTGAA--CTCCACTGCAAAA---TCAGTTCCTCTGATGATCCGGGATCTGCGAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V33.0.8 TAAACTCACTTATGGTGAAGGAACTGTTGAGGCTGATTTTAAG-------------GAT---AGATTTTCTGCA-ACACTGAA--CTCCACAGCAAAA---TCGGTTCCTCTGATGATCAAGGATGCACAATTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V33.0.9 TCAATACAG---------------CACAGAGGCTGACTTTAAA-------------GAA---AGATTTTCTGCA-ACACTGGA--CTCCAAAGAAAAA---TCATTTCCACTGACAATCAAGAATCTGCGAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAAGCC---------------- V33.0.13 TCAATACAG---------------CACTGAGGCTGACTTTAAG-------------GAG---AGATTTTCTGCA-ACACTGGA--CTCCACAGAAAAA---TCATTTCCACTGACAATCAAGAATCTGCATGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V33.0.10 TCAATACAG---------------CGCTGAGGCTGATTTTACA-------------AAG---AGATTTTCTGCA-ACACTGGA--CTCCAAAGAAAAA---TCATTTCCACTGACAATCAAGAATCTGCAAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTTTGAGGCC---------------- V33.0.11 TCAATACAG---------------CGCTGAGGCTGATTTTAAA-------------GAG---AGATTTTCTGCA-ACACTGGA--CTCCAAAGAAAAA---TCATTTCCACTGACAATCAAGAATCTGCGAGTATCAGACTCTG--CTGTGTATTA-CTGTGCTCTGAGGCC---------------- V33.0.12 TCAATACAG---------------CGCTGAGGCTGA---TAAG-------------AAG---AGATTCTCAGCA-ACACTGAA--CTCCAAAGAAAAA---TCATTTCCACTGCTGATCCAGGATCTACAAGTATCAGACTCTG--CTGTGTATTA-CTGTGCCCTGGAGCC---------------- V33.0.3 TGAATTTACCATTGGTGAAGGAACTACTGAGGATGAATTTAAA-------------GGG---AGATTTTCAGCA-ACACTGAA--TTCAACATTAAAA---TCTGTTCCTCTGCTGATCCAGGATCTGCGAGCGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTAAGGCC---------------- V33.0.4 TGCATTTACAGTTGGCAAAGGAACTACTGAGGATGAGTTTAAA-------------GAG---AGATTTAACGCA-ACACTGGA--TAAAACATTAAGA---ACGGTTCCTCTGCTGATCCAGGATCTGCAAGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTTTGCAGCC---------------- V33.0.1 TGAGTTTTCCATCGGGAAAGGAAAAGCAGAGCCTGAATTTAAG-------------GAC---AGATTCTCAGCA-AAGTTGGA--CTCCAAATTAGAA---ACAGTTCCTCTGACAATCAAGGATGTGCGTGTGTCAGACTCTG--CTGTGTATTA-CTGTGCTCTGCAGCC---------------- V18.0 AAGGCACAATTTGGGAAATGGAAGTAATTCCAAAGACTTCCCC-------------AACAACAGATATCAGTCC-CGGTTGGA--TTCTGCTATGGGA---CAAGTTACTCTGACTGTACGAAACCTTCTTCTGTCTGATTCTT--CAGTTTACTT-CTGTGCTTTGAGGCC---------------- V27.0 AAGATATAAATTTGGAGATGGAGATGAAGCTACTGAGTT---C-------------AAGGGACGATTTGGGTCC-AGACTCGA--TTCTGCTTCAAAA---TCAGTTCCTCTGACGATTCAACATCCGCATGCGTCTGATTCTG--CTGTTTACTT-CTGTGCTCTGCTGCC----------------

Supplemental Figure 3: Multiple sequence alignment of nucleotide sequences used for Figure 2 tree. Mega 6.0 program was used and Clustal W methodology was employed for alignment. Highlighted nucleotide sequences represent coding sequences for the conserved C, WYRQ, YxC sequences respectively.

* **** *** V30.1.3 ---CDSQDKVDQH--TKTQSAFEGDTVTIDCTYQTSYTT---PTLFWYQQKVN-GVPKHMLNKIS-TSG-----HTEEEFKE-RFHATLSKTS--------VPLTIKD--LRVSDS------AVYYCALR---- V30.1.4 ---CDSQDKVDQH--TKTQSAFEGDTVTIDCTYQTSSTG---PTLFWYQQKVN-GVPKYMLNKIT-TTG-----HTEEEFKK-RFNATLSKTS--------VPLTIKD--LRVSDS------AVYYCALR---- V30.1.1 ---CDSQDKVDQD--TKTQSAFEGDTVTIDCTYQTS-TN---PTLFWYQQKVK-GVPKHMLNKITNTTG-----HIEEEFNE-RFHATLSKTS--------VPLTIKD--LRVSDC------YVLLCSEA---- V30.1.6 ---CDSQDKVDQH--TKTQSAFEGDTVTTDCTYQTSSTG---PTLFWYQQKVS-GVPKHMLNKFS-TTG-----NSEEELKKKRFHATLSKTS--------VPLTIQN--LRVQDS------AVYYCALK---- V30.1.7 ---CDSQDKVDQH--IKTQSAFEGDTVTIDCTYQTSYTT---PTLFWYQQKVN-EVPKHMLNKFS-TTG-----NSEEEFKK-RFNAALSKTS--------VPLTIKD--LHVSDS------AVYYCALK---- V30.1.5 --GCDSQDKVDQH--TKTQSAFEGDTVTIDCTYQTSYTG---PTLFWYQQKIK-SPDIC*IKSLT-HQD-----TLRMN*RR-DFMQLSAKLT--------*LLYFIT--LKKQTC------HKIIPTES---- V10.0.1 ----TLAQSIRPL--QSDVYITERTPVKLSC--KYDGS---VYSLHWYPQYPG-AKPEFLLLVLKSAT-KS---VTYADPKIPGMDGEMSISE------QQVDLEISS--AAVSDS------AVYYCALQ---- V10.0.4 ----TLAQSIRPL--QSDVYITERTPVKLSC--KYDGS---VYSLHWYRQYSG-AKPEFLLLVTESTS-KI---VTYADPKIPGMDGEMRMSE------KQVDLEISS--AAVSDS------AVYYCALQ---- V10.0.3 ----TLAQSIRPL--QGDVHITERTPVRLSC--KYDGS---AYSLHWYRQYPG-AKPEFLLLVRESAK-KS---VTYADPKIPGMDGEMSMSE------KQVDLEIFS--AAVSDS------AVYYCALE---- V10.0.2 ----TLAQSIRPL--QSDVHITEKTPVRLSC--KYDGS---ANSLHWYRQHLG-SKPEFLLLVMESVI-RH---VTYADPKIPGMDGEMSMSE------KQVDLEIFS--ATISDS------AVYYCALQ---- V10.0.5 ---ECFAQNITPL--QGTTQVTEGKTVTLSC--KYDVS---VQSLLWYRQYPG-SGLEFLLLVIESPT-KT---VIHAKPPIPRVDGEMSMSD------KRVNLEISS--AEVTDS------ALYYCALV---- V11.0.1 ---QSFGQSITAL--QSTKQAAESETVILSC--KYDTR---ADNLQWYRHYPG-STPEYLLLVYES---NT---VFHAEPPFPRLNASVNKVE------KQVNLTISS--AAVTDS------AVYYCALQ---- V11.0.2 ---QSFGQSITAL--QSTKQAAESETVSLSC--KYDTQ---ANNLQWYRQYPG-STPEYLLLVYES---NT---VFHAEPPFPRLDASVNKVE------KQVNLTISS--AAVTDS------AVYYCALQ---- V11.0.3 ---QSFGQSITAL--QTTKQAVESETVILSC--KYDTQ---ADSLHWYRQYPG-STPEYLLLVYES---NT---VFHAEPPFPRLNASVNKVE------KQVNLTISS--AAVTDS------ALYYCALQ---- V17.0 ---TSRQDSITPT--SPAVHVKEKEAARISCEYKYTVS---MNNLQWYRQYPN-AKPEFLVLIMES---GQ---NQTATPPHPRLTTSVNKDK------KLVNLEIFP--ADVKDS------AVYYCALQ---- V38.0.1 ---ETFAQSITPL--ENKTLKSEGETVTLSC--KYEG---VVNNLHWYRQYPG-FRPKFLAYIYEHG--------ATSKPLPPRLLPKVDKNS------KRVSLEISE--AEVTDS------ALYYCALT---- V38.0.2 ---ETFAQSITPL--ENKTLKSEGETVTLSC--KYEG---VVNNLHWYRQYPG-FRPEFLAYIYEHG--------ATSKPLPPRLLPKVDKNS------KRVSLEISE--AEVTDS------ALYYCALT---- V38.0.3 ---DTFAQTITPL--ENKIVKSEGETASLSC--KYEG---SVTNLHWYRQYPG-SRPEFLAYIYEHG--------ATSKPLPPRLLPKVDKNS------KRVSLEISE--AEVTDS------ALYYCALT---- V14.0 ---IIQTNMITSH--DPQKQAFEGEYVKLAC--NYSG---APDNLHWYRQYPG-STPMFLLYIYESG--------LKSDNIPERLTPKINKTK------KVVDLEISS--ATVSDS------AVYYCALK---- V5.0.1 ---DSLGDIITSR--GSEKAAQDGTNVTLSC--NYTG---AVYNLLWYRQYQR-FKPELLLSITESG----E--AVKADPSAHWLSATVHKQS------KLTELEISP--AKVTDS------AVYYCALK---- V5.0.2 ---EVIGEVITSR--GSKKEAQDGTKVTLSC--NYTG---VVYNLLWYRQYQR-FKPELLLSITESG----E--AVKADPSSHWLSATVHKQS------KLTELEISP--AKVTDS------AVYYCALK---- V3.0.2 ---DAGEEVISSL--STEQIVLAGKNATLSC--KYNG---NVYNLQWYRQYPG-SKPENIIFHTESK--------NQSDFK-LRLLAVGEKGM------KRMNLSIFH--TEMQDS------ALYYCALQ---- V3.0.3 ---TAGEEVISSL--STEQIVLAGKNATLSC--KYNG---NVYSLQWYRQYPG-SKPENIIYHTESN--------KQSHSR-LRLLAVTEKGM------KRMNLSIFH--TEMEDS------ALYYCALE---- V3.0.1 ---NAGKEVISSL--STEQIVLAGKNATLSC--KYNS---NIRNLQWYRQYPG-SKPENIIFHTESN--------NQSYSR-LRLLAVAEKEI------KRMNLSIFH--TEMQDS------ALYYCALE---- V3.0.4 ---NSGEEVISTL--PTNQYIIEEKNATLSC--NYSG---YVQNLQWYRQYPG-SKPENIIFYTESN--------NQSDSK-LRLLAVADKGQ------KLMNLSMYF--TKVEDS------ALYYCALQ---- V7.0 -NVDADGHMINPL--SLETHGPVGHNVTLSC--NYSG---RVHTLQWFRQYPG-SRIEFLIFATELN--------DQSNAA-LRISNVVDK-Q------KSMSLSIFH--AEIQDS------ATYYCALE---- V8.0 ---IADAYTIESL--SAEKQVSVGGNAVISC--KYSG---IGPSFQWYRQYPG-SRPEYLIFNTETG--------AGSEPT-LRLTSMAKKAL------TQVDLEISS--TEVKDS------AMYYCALQ---- V34.0.3 ---ISIADEIHPL--STDKHVAEGQNVTLSC--NYSTSTGTVNSLQWYRQYPG-AKPEFLLSLNEYS--------NSSKPD-LRLVSNAVKAL------KRVDLLLSS--AAVSDS------AVYYCALQ---- V34.0.4 ---TAIADEIQPL--SSDKRVAKGENVTLSC--NYST-TVTVDSLQWYRQYSG-AKPEFLLSLNEYS--------NSSKLD-LRLVSNAVKAL------KRVDLLLSS--AAVSDS------AVYYCALQ---- V34.0.2 ---TSSADEIHPL--FLDKQVAEGQNVTLSC--NYSISIGILRSLQWYRQYSG-AKPEFLLMLNEYY--------NKSKLD-LRLFSNAVKAL------KRVDLLLSS--AAVSDS------AVYYCALQ---- V34.0.1 ---MSFSDEIHPL--SSDKHVAEGQNVTLCC--NYRASTGNVDSLLWYRQYPG-ANPEYLLLLNEYS--------NSTKSY-LRLFSNAVKAL------KRVDLLLSS--AAVSDS------AVYYCALR---- V34.0.5 ---NLFGDTIEPL--FPDRHGSEGENVTFSC--NYSLTSGTSVNLLWYRQYSG-AKPEYLLSVTEYS--------NSSESD-LRLFSNAVKAL------KRVDLLLSS--AAVSDS------AVYYCALR---- V34.0.6 ---NSFGDEIQSL--VKHEHGSENENVTFSC--KYRLTSGTNVNFQRYRQYSG-AKPEYLLSVTEYS--------NSSESD-LRLFSNAVKAL------KRVDLLLSS--AAVSDS------AVYYCALQ---- V4.0.1 ---NAGEEVIRPL--FKEKHVLEGDVVTLIC--SYSG---SVQNLQWYHQFPG-SKPENIFLYFEN---------KPQTE--GRMTAASDKAV------KHMNLTITS--TQLTDS------AVYFCALV---- V4.0.3 ---NAGEEVLRPL--FKEKHVLEGDVVTLIC--SYSG---NVLNLQLYHQFPG-SKPENIFLYFEN---------KPQTE--GRMTAASDKAV------KHMNLTITS--TKVKDS------AVYYCALQ---- V4.0.2 ----SLQWTLFIL--FKEKHVLEGDVVTLIC--SYSG---NVQNLQWYHQFPG-SKPENIFLYFEN---------MFTTK--NRMIAASDKAV------KHMNLTISS--AEITDS------AVYYCALQ---- V9.0.1 --KAFFSNAIRPL--SAEQQVLEGGKVILSC--AYNGS--NIKSLQWYKQHHS-PAPEYLLQTFENA--------APEQK--DRLLAKAQKDK------KQLDLEISK--AEMSDS------AVYYCALV---- V9.0.2 ---GIFANSVTPW--STKQQVLEGENVTLSC--NYSGS--NIQSLQWYRKYPSSTAPEYILQTFESA--------EPAQQ--GRHLAKAQKNK------KQLDLLISK--AEMTDS------AMYYCALV---- V6.0.1 ---HTEADVIKPL--SLDENSHVGDAVTLSC--SYKDYTGNMQNLQWYRQFPR-SKPEFILYIFESG--------TMSKDKPDRFSAQVNKGT------KQVDLNISP--AVETDS------AMYYCALV---- V6.0.2 ---HTEADVIKPL--SLDKNCHVGDAVTLSC--SYKDYTGTVQNLQWYRQFPR-SKPEFILYIFESG--------TMSKDKPDRFSAQVNKDT------KQVDLNISS--AVETDS------AMYYCALV---- V6.0.4 ---HTEADVIKPL--SLDENSHVGDAVTLSC--SYKDYTGTVNGLQWYRQLPR-SKPEFILYIFESG--------TLSEDKPDRFSAQVNKDT------KQVDLNISS--AVETDS------AMYYCALV---- V6.0.3 --QHTEADVIKPL--SLDENKHVDDTFTLSC--SYKDYTDTVRNLQWYRQLPR-SKPEFILYIFESG--------TMSKDKPDRFSAQVNKDS------KQVDLNISS--ALETDS------AMYYCALV---- V12.0.2 ---DTSGEGIAHH--SSSEFFINGHSTTLSS--NYNGS-YSSDSLLWY*QYSS-SKPQFLYLVSEA----KL--EQPADPPIPGLPAKLNEEN------NLVYLEISS--AALSDS------AVYYCALT---- V13.0 ---DAFGEGIAPV--SGYKTVPEEKTTTLSC--IYNGS-AATDALQWYRQYPR-SRPDFLFLVNEA----AF--KQPANPPIPGISARLNEEK------NCVYLDITS--TAISDS------AVYYCALQ---- V2.0.1 ---VVNGNVITPA--RTSVVLTEGSNITLSC--TFNTS-PYR--LLWYRQTPG-SRPEFLLLIVRST---KT--TIEASPPQPHMSINLSE--------KLVDLEISS--VAVSDS------AVYYCALE---- V2.0.3 ---DSNGNVIRPE--KTSVVLTEGSNITLSC--TFNIT-PYS--LHWYRQMPG-SRPEFLLLIVRST---KT--KVAASPPQPHMSINLSE--------KRVDLEISS--AAVSDS------AVYYCALE---- V2.0.4 ---NANENSIRPD--EEFVSLNEGSNTTLSC--TFDGS-AYR--LHWYRQKTG-SLLEFLVLIDKST---KV--VVTASPPHPHVSINLHD--------NRVDLEITP--AAVSDS------AVYYCALQ---- V2.0.2 ---DSNGDIIRPE--QTSVVVTDGSSITLSC--TYDES-AYS--LHWYRQTPG-SRPEFLLLIDESS---KY--VTKAEQPHPHMSIQLQNN-------KRVNLILSF--AAVSDS------AVYYCALQ---- V1.0 ---ETTGDKIKPD--NDTAQLMENSNITLSC--TYEGS-IDS--LYWFQQKIG-SRLEFLIMIDEAS---EY--VTNAIPPHPRLSVQLHKAQ------KRVSLEMSS--AAVTDS------ALYFCALR---- V24.0.1 ---NLAKDVITPY--SDEIFASETESVKFSC--NYTGS---VDGLHWYRQYPD-SPPHFLILDYYGS-------ITHANPPIAGISIKHRKDN------SSVDLEISS--VAVSDS------AVYYCALQ---- V24.0.2 ---NRAKDAITPY--RDAVFASESERVKLSC--NYTGS---VDSLHWYRQYPG-SRPHFLILDYSGF-------VTHANPPFDGISIKHRKEN------SSVDLLISS--AAVSDS------AVYYCALR---- V24.0.4 ---SSATEAITPY--RAYEFAFKDDRVILSC--NYTGS---ARGLHWYRQYPG-LPPHFLILDYYGA-------VTHAKPPIAGISIKHRKDD------SSVDLEISS--AAVSDS------ALYYCALE---- V24.0.3 ---SRASEVITPK--TDREFAVEGDNVTLSC--NYSGS---VRSVHWYRKYSG-SPPQFLILEYSGV-------ITPADPPVPGISINHRKKQ------SHVDLLLSS--AAVSDS------AVYYCALQ---- V24.0.5 ---SRALEAIDPK--TDREFAVEGDNVTLSC--NYSGS---VRGVHWYRQYAA-SPPQFLILEDSGV-------TTPANPPVPGISINHRKAE------KHVDLLLSS--AAVSDS------AVYYCALR---- V28.0 --VSSAEEIIFPN--NKTVIAKADEKVLLSC--SYTGT---VYDLYWYRQSPG-SAPQFLIMESSGV-------IIHATPQVPGIKIINKKNT------KHVELEIFS--ASVEHS------AVYYCAAR---- V36.2.10 ---AVFGNVIKPI--NTDVFAEEGSNVTLSC--SFTASG-VTDYLHWYRQYGR-SKPEFLVLTYSSAT------KAAQSDIDTRFTTNITKKE-------HVDLLLSS--AAVSDS------AVYYCALR---- V36.2.2 ---AVFGNVIKPI--NTDVFTEERSNVTLSC--SFTASG-GSDYLHWYRQYGG-SKPEFLVLIYGSAT------KAAQSDIDTRFTANITKKE-------HVDLLLSS--AAVSDS------AVYYCALQ---- V36.2.8 --PAVFGNVIKPK--QTDVFADEESNVTLSC--SFTASG-GSDYLHWYRQYGR-SKPEFLVLTYRTAT------KAAQSDIDTRFTTNVTKKE-------HVDLLLSS--AAVSDS------AVYYCALQ---- V36.2.11 FSAAVFGNVIRPI--NTDVFAEEGSNVTLSC--SFTASG-GTDYLHWYRQYGG-SKPEFLVLTYSSAK------KSKESDIDPKFSANVSKSE-------HVDLLLSS--AAVSDSGPCIIRSFDYSFYYG--- V36.2.9 TPH*KIKCVLDLLVCKTDVFVEEGSNVTLSC--SFTASG-GSDDLHWYRQYGG-SKPEFLVLTYRTAK------EAEKSKVDPRFIANVPKSE-------HVDLLLSS--AAVSDS------AVYYCALR---- V36.2.1 ---SVYGNVIIPD--ETEAVHAKGSDVKLSC--SFLASG-YTDYVYWYRQYRR-SKPEYLVQIISNSN------KAQQSLVDPRFTAYIENRDK-----IHVDLLLSS--AAVSDS------AVYYCALR---- V36.2.12 ---TVFGNTIKPD--ETNVVAQKDSDVKLSC--SYSST--TTDTLYWYRQYER-SKPEFLVLIYSSAK------AAEKSKVDPRFFVKVEKRQQ-----IHVDLLLSS--TAVSDS------AVYYCALQ---- V36.2.5 ---SVLGNDIKPN--ITDVVYEEGSDGTIFC--SHSPG--AGDYIYWYRQYGG-SKPEFLVLIFGSTK------DTETSKVDPRFSVTVEKKER-----NHADLLLSS--AAVSDS------AVYYCALQ---- V36.2.7 ---SVFGNDIKPN--STD-VYEEGSDGTIFC--SHSPG--AGDYIYWYRQYGG-SKPEFLVFTFGSVK------DAETSKVDPRFSVKIEKKEQ-----NHADLKISS--AAVSDS------AVYYCALR---- V36.2.6 ---SVFGSDIKSN--ITDVVYKEGSDGTIFC--SHYPG--TGDNIFWYRQYGG-SKPEFLLATYGSAK------DTETSKVDPRFSVKVEKKER-----NHADLKISS--AAVSDS------AVYYCALR---- V36.2.4 ---SVFGNDIKPN--STEVVLKEGSDGTIFC--SHSPG--TGDYIHWYRQYGG-SKPEFLVLIYGTAN------KGETSKVDPRFSVKVEKKER-----NHADLKISS--AAVSDS------AVYYCALQ----

* **** *** V36.2.3 ---SVSGNLIKPI--QTDVFADEGSNVTLSC--SYSSA----DYLFWYRQYPR-GAPEFLVSIFEGER------NSKSSDVDPRLSAKLTKSEH-----NHADLMLSC--AAVSDS------AVYYCALR---- V36.1.2 ---ATYADEIKPV--KTEVFADEGSNVTLSC--TYSSA----NNLQWYRQYPG-SAPQYLVLIPDSSK------EDWKSNVDDRFTARVTKNKE-----KRVDLLLSS--AAVSDS------AVYYCALE---- V36.1.3 ---AIYADEIKPV--KSEEFADGGSNVTLSC--TYSSA----YYLYWYRQYPG-SAPQHIVLIPESRK------DDTKSDVDHRFIARVTKDKE-----KRVDLLLSS--AAVSDS------AVYYCALE---- V36.1.1 ---ATYGDEIKPV--KTEKFADKGSSVKLSC--TYSSA----NTLFWYRQYPG-SAPQYLVLIPEGTK------DDWKSDVDKRFTARITKDKE-----KHVDLLLSS--AAVSDS------AVYYCALR---- V36.1.6 ---VAYGNEIKPV--KTEEFAKEDSSVTLSC--SYSSA----RTLFWYRQYPG-LAPQFLVFFIHGTS------EAKESGVDPRFTAKPNGEKQ-----SHVDLLLSS--AAVSDS------ALYYCAME---- V37.0 ---VIFGDEISPD--SSEVFAAVASKVTLSC--RYSSA----YNLQWYRHYPG-SAPQNLVLISEGLT------ETKISEVDSRFTTKLRKENQAGKEIKRVDLLLSS--AAVSDS------AVYYCALR---- V36.1.4 ---ASNGDTITPD--KTEEFADEDSNLTLSC--SYSSA----WNLLWYRQYPG-SALEYLVLTVDTVN------EARTSDVDVRFATKTRKENA-----NHVDLEISS--AAVSDS------AVYYCALK---- V36.1.5 ---ASSEDTITAD--KTEEFADDGSNVTLSC--TFSSA----WSLLWYRQYPG-SAPEFLVNIAERSK------KVERSNTDHRFTTKIRIEKE-----KHVDLLLSP--AAVSDS------AVYYCALTSM-- V19.0 ---GKSDSPITSD--QTEVFPRENANVTLSC--SYSSA----ISLHWYRHLPR-SAPEFLLLVLQTTGRV-----QTAQNLDSRFEAMLNEKKN------KVHLIISS--VKVTDS------ALYYCAME---- V20.0 ---VTHADAITPD--RPEVNVNETESITLSC--QYSSA----FSLHWYRQHDA-SGPQFLMLIQQSTG-------QMIQRSDDRLSGKLNKEKN------RVDLQISS--STIKDS------ALYFCALQ---- V25.1.2 ---VSFGNVITSV--ETRLLHSEGDVISVSC--SYSSA----RTLFWYRQNPG-TVPQFLFVILESNGDVLQ--KSEAVK-DPRFSAKVNEKKT------HVFLEISS--AKVTDS------AIYYCALE---- V25.1.3 ---VSFGNVITSV--ETRLIHTEGDVISVSC--SYSSA----RTLFWYFQNPG-SVPQFLFIILHSNGEVLQ--KSEAVK-DPRFSAKVNEKKT------HVFLEISS--AKVTDS------AIYYCALE---- V25.1.1 ---DVLGNVIIPV--HSEVYKTEKETISLSC--NYSSA----SSLYWYRQYPK-STPEFLFVILHATGKVLQ--KSDIVDQDPRFSGKLNEEKT------HTSLNISS--AKVTDS------AMYFCAME---- V25.1.4 ---VTFGNDITPV--QTEVFGAEMDNITVSC--KYSSA----VSLQWYRQYPG-SAPDFLLTIHR-TGSVSR--SSTIVNEDPWYYGKLNKEKT------HVFLEIST--AKVSDS------AVYYCALQ---- V15.0 ---VSCGDVITPA--QTEVYGKSGNKVTLSC--NYSTA----LSLYWYRQYPG-SNPHCLLLILT------K--SETSENMQPGMSVRLHKEKA------SMDLKISS--AKLTDS------AVYYCALK---- V23.2.8 --GVTNADQIGS-NIGENVFSIEGETATLNCSYATSSR---NVRLYWFRQYHN-KEPQYLIWKDCRSYS------NTVTSADPRVEPKTSETT--------AELIIQS--VTLSDS------ALYYCALR---- V23.2.4 ---VTNADQIGP-NKGENAISKEGETATMSCSYDTSSR---YVRLYWYRQYTN-KELQYLIWKDARSWS------GVATPDDPRIRSTTSETT--------TELIITG--VTLSDS------ALYYCALRVG-- V23.2.6 ---V*RMQIRLD-QIRVKIPSVKRE--RLIC*-----------PLLVYRQYTN-KELQYLIWKEARSWS------ATGTPDDPRIRASTSETT--------TELIITG--VTLSDS------ALYYCALIVG-- V23.2.1 ---VMTADQIRP-NKEAFTVKEE-ETVTFSCSYETSSS---YVWLYWYRQHLN-GEPQYLIFKPAKSAS------VSGDPVDRRFQSSTSDSS--------TELTISG--ATLSDS------ALYYCALRVG-- V23.2.2 ---VMTADQISP-NKEALTVKEE-ETVTFSCSYDTSSS---YVRLYWYRQYLN-GEPQYLLFKAARSSS------GGGRPDNPRFKSTTSDSS--------TELTISG--VTLSDS------ALYYCALRVG-- V23.2.3 ---VMTADQIRP-NKDVTVIKEE-ETVTLSCSYE-SSS---TVRLYWYRQYLN-GEPQFLVSSAQSSSG--------------KFQTAKSRSS--------TELTISG--VTLSDS------ALYYCALRVL-- V23.2.5 ---VMTADQIRP-NKDSTVIKEE-ETVTLSCSYE-SSN---YVRLYWYRQYLN-GEPQYLIHSAGSNIG--------------RFQIAKSDSS--------TELTISG--VTLSDS------ALYYCALRVG-- V23.2.7 ---MMSVDKIGP-DKDTNVISKEGETVTLSCSYDTQSS---YVRLYWYRQYAD-REPQYLIWKEARSLR------GAGNSFDSRFQSTTSESS--------TELTVTG--VSLSVS------ALYYCALFVG-- V23.3.1 ---MVCGDSIEP-DKGKEKTSEEIENVKLSCSYSTSSS---YVCLYWYRQYLN-GEPQYLVHRYARSWG------NDEYKSDDRFQSATSESS--------TELTITN--VHLSDS------ALYYCALRVR-- V23.3.5 ---MVCGDSIEP-DKGTKKKSEETESVKLSCSYSTSGE---DVLLYWYRQYLN-GETQYLLRRYARSLG------NNEYKSDDRFHSATSSSS--------TELTITN--VRLSDS------ALYYCALRVI-- V23.3.4 ---MVCEDSIEP-DKGTEKTSEETENIKLSCLYTTSSD---YVWLYWFRQYLK-GEPQYLLYKGARSY-------STHHKSDDRFESATSRSS--------TELTITN--LRLSDS------ALYYCALRVR-- V23.3.7 ---MVCGDSIEP-DKGTEKNSEETENVKLSCSYSTSSE---YVYLYWYRQYLT-GELQYLLRKGAQSY-------LTHHTSDKRFESATSRSS--------TQLTIND--VSLSDS------ALYYCALSVG-- V23.3.6 ---MVCGDSIGP-DKGTEKT-EETETVKLRCSYSTNSE---YVWLYWYKRSLN-KEPQYLLFESARSA-------SDSHSSDDRFHSATSRSS--------TELTINN--VRLSDS------ALYYCALRVG-- V23.3.2 ---MVCGDSIGP-DERTENTSEETKTVKLSCSYSASIE---YVVLYWYRQYKH-GEPQYLLRRYARSYN------DYEATSDDRFQSDSSYTS--------TELTIKD--ARLSDS------ALYYCALIVG-- V23.3.8 ---MVCGDSIEP-NKGMEKTIKETEPVQLSCAYTTATSR-SYVYLYWFRQSFN-GEPQYLLNKNAQTA-------SDSHTSDDRFQTTITQTA--------TELTITD--VRLSDS------ALYYCALRVG-- V23.3.3 ---MVFGDNIEP-EE-KDVMTKEREAVKLACSYSTTNN---RVRLYWYRQNPN-AELLLLTYKGARSL-------SAKHSSNDRFQSTTSDSS--------TELTITD--VRLSDS------ALYYCALRVG-- V40.0 ---VTAVDKIEP-NSGSKVIRTERESVKLSCSYDSNNE---YVRLYWYRRHSS-GELQYLLYKGARSN-------SAKDTSDARFESSTSRIS--------TDLIIKT--AALSDS------ALYYCALRVE-- V23.0.1 ----GYTDRIGPGEGDTNIISMEGESVTLSCTYETNSN---DIWLYWYRQFSN-REPEYILFKGARSRS------E-EDLP-DGFQSRTSDST--------TEITINS--VRLSDS------ALYYCALEV--- V23.0.2 ------TDRIGP-EGETNILQEEGQSVTLSCTYETNSN---TIYLYWYRQYPK-TKPEFILYKGARSWS------S-EDIP-DSYESATSHTS--------TKLIIKS--VSLSDS------ALYYCALLVR-- V23.0.3 ----ALSDSIGPNEKETNVMSREGESMRLSCSFESSSN---YVLLYWFRQNPD-GELQYLLYKGARSWS------SKEDHSEDRFQSIAAYSS--------TELNTAE--AALSDS------ALYYCAVKSRLY V23.1.10 ----VFADSIRP-DKDT-MTSYEGESVTLKCSYDTSSN---NVLLYLYKQNVN-TEPQYLLWKKARSYN------RDEDIPDSRFDSITSQTS--------TELIIKS--VSLSDS------ALYHCALTVG-- V23.1.6 ----VFADSILP-DKDSNMTSNEGESVTLKCSYDTSSN---NVLLYWYRQYAN-TEPQYLLWKGARSNS------NYKKIPGSQFDSITSQTS--------TELIIKS--VSLSDS------ALYYCALRVG-- V23.1.7 ----VFADSIRP-DKDTNIIRSEGESVTLRCSYETNSN---YIRLYWYRQYVN-TEPQYLLWKGARSDS------DYKNIPDSRFDSITSQTS--------TELIIKS--VSLSDS------ALYYCALRVR-- V23.1.3 ----VFADSIKPKGKDK-IIRSEGESVTLSCEYETSSN---NVMLYWYRQYTN-REPQFLLRKGARSRN------GLDDIPDRQFDSTSSQTS--------TELTNTG--VSLSDS------ALYYCAIRVG-- V23.1.5 ----VFSDSIGPKDEDKHIIRREGESVTLSCSYETQSN---NVWLYWYRQYTN-KEPQYLLLKGARSNS------RSEDRPDHHFQSSTGQTT--------TELTIDK--AALSDS------ALYYCALRVG-- V23.1.1 ----VFADSLQPKDKDK--IISEGQSVTLSCTYDTSSS---YVYLYWYKHNIK-GEPKFVLCKGARSRS------DRQYIPNRQFDSITSQTS--------TDLIIKS--VTLSDS------ALYYCALLVE-- V23.1.8 ----VFPNNIWPKDKEV-IVSNEGESVTLTCSYSSGSN---DVWIYWYKYLSN-TEPKFLLWKGARAQN-------MQHIPDQRFESTTYQTS--------TNLFIKS--VSLSDS------ALYYCALRVV-- V23.1.4 ----VFADSIGPNDKETSVSSNEGESVTLSCSYDAQSN---NIRLYWYRQNSN-REPEYLLWKGARSYG------GEDKT-DSRFNSKTSRTA--------TELTISA--VTLSDS------ALYYCALRVL-- V23.1.9 ----VLPDSIGPTDKDAIKTRKEGESVTLSCSYDTQSN---NVYLFWYRQFTN-REPEYLLIKGARSYG------GEDKI-DSRFRPTTSQTS--------TELTIPG--VTLSDS------ALYYCALRVV-- V23.1.2 ----VLSDSIQPTDKKTNTDTKEEEHVTLSCSYDTRSE---YVYLYWYRQYPN-REPEYLLRKGARSWS------SYNNMPDPQFQATSSQTS--------TELTITA--VTLSDS------ALYYCALRVE-- V26.0 ----VFADSIAPKKEGRNKTRKEGESVTLSCTFNRSDT---AISLYWYMQYPN-RELQYLLYKGHRSSE------E-GIVTDSRYKSTVSPTS--------TSLTINS--VNLSDS------ALYYCALKKG-- V16.0 ----AFGDTIGP--DEPLIFRSETDRVTLSCSYQTNSE---HVDLYWYRHYPK-SEPQYLLWKGARARD------GEKHMPDSHFESSTSRTS--------TELIIKV--AALTDS------ALYYCALWK--- V22.0 ---AMCQDQIGP--SRTSVTSTEGDSVNLECSYDSSIN---NVFLYWYRKYSN-AQPQYILLKGGRSQT------YEDIPDRAKFGSATSQKS--------TTLTIKS--VTVSDS------ALYYCALRVG-- V21.0 ---ITFCAHIAP--NTKFVIGREGESATLSCLYESSSS---HVLLFWGRILPK-QAPEFLLLKGARSET------QFQS-VDPRYDSATSKTA--------TFLIINN--LTLEDK------ASYYCVLEE--- V27.0 ---SRAQ-DSVYQPKTLQSANEEDAAVTLDCKYSTSSSN---PTLLWYRQDDY-GSPKYILLRYKFGDG-----DEATEF-KGRFGSRLDSAS------KSVPLTIQH--PHASDS------AVYFCALL---- V41.0 ---CKAQ-DNVIQPKQVMTAY-EDGTITLECTYKASSTQY--PNLLWYQQMTN-GSPKYMLMRLS-GSS-----DTNEEF-KQRFNAELNTSA------LSIPLTIKD--LRASDS------AVYYCALK---- V32.0.2 ---CRSE-DRVDQPDKQLTEA-EGGSVTLQCKYKTDSVQ---ENLFWYIQRTN-DLPKYILRRSKYG-G-----ENGTEF-QERFHSELST--------NSVPLTIRD--LHVSDS------AVYYCALK---- V32.0.4 ---CRSE-DRVDQPDKQLTEA-EGGSVTLQCKYKTDSTQ---EDLFWYIQRIN-DQPKYILRRSKYG-G-----ENGTEF-QERFYSELST--------NFVPLTIRD--LHVSDS------AVYYCALR---- V32.0.6 ---FRSE-DRVDQPEKEITVA-EGGSVTLQCKYESYSVQ---KDIFWYVQRTN-DFPKYILRRSNFG-G-----ENGTEF-QERFHSELSL--------DSVPLLIRD--LRVSDS------AVYYCALR---- V32.0.3 ---CRSE-EKVDQPDKHMTEV-EGKSVTLRCTFTTTSNQ---PDLFWYIQRAN-DHPKYILRRDKYGAG-----ENGTEF-QERFHSEFLT--------DSVPLLIRD--LHVSDS------AVYYCALR---- V32.0.5 ---CRSE-EKVDQPDKHMTEV-EGKSVTLRCTFTTTSNQ---PDLFWYIQKAN-DHPKYILRRDKYGAG-----ENGTEF-QERFHSELLT--------DSVPLLIRD--LHVSDS------AVYYCALR---- V31.0 ---CHSD-DTVDQPLKHVTAL-EGISVTLDCKYTTSSTS---QELFWYIQRAD-GSLK--LQRNSYGGG-----VNGTEF-KERFYSEVKTS-------KSVPLLIQD--TCVCDS------AVYYCALR---- V32.0.1 ---CRP--DSVNQPDTHVTKS-EDGAVTLQCKYTATSTAT--PELYWYIQREH-EIPKYILRKNKYE-G-----ENGTEF-HKRFHVELTT--------DSVPLLIKD--LRVSDS------AVYYCALK---- V29.0 ---CRAQ-DSVTQTATVQTAT-EGQAVTIECTYK-TNAF---PTLYWYQYKAN-QFPKYMLKRYAGSSD-----EDDNFK--DRFNANLNSSS------TSVPLTITD--VRVSDS------AVYYCALQ---- V30.0.6 ---IESQ-DNVEQKARVQTAV-EGEAVEIDCTYQ-TQLT---PTLFWYQQKTN-TIPKYMLNRVGKTGG-----EDKEFK--ERFTANLNTFS------KSVPLMIRD--LHVSDS------AVYYCALQ---- V35.0 ---CKCK-DSVHQKR-VETAV-EGTDLFISCSYTSTDPI---PTLFWYQQKVN-EFPKYMLSTFE---S-----KDDEFK--KRFNARLNTSS------TSAPLLIQD--LRVSDS------AVYYCALK---- V30.0.5 ---FVSP-DSVEQKTRVQTAV-EGESVTINCTYSTTDTF---PYLFWYQQQVN-GIPQYMLKKFPGSGE-----IENQFK--KRFDADLDSST------TSVLLRIKE--VHVSDS------AVYYCALK---- V39.0 ---RVSL-DKVEQNSRVETVV-EDEDVIINCQYETTDTL---PYLFWYQQKVN-RFPEYMLNRFSSD--------EEQFK--DRFSSELNTSS------KSFPLKIQD--VRVSDS------AVYYCALR---- V30.0.2 ---CDCA-ESVDQNTRVETSV-EEGSVSISCTYQTSDPS---PNLFWYQQKPN-TIPKHMMTIITTTVQ-----NDKDFE-EERFGAKHDKTL------KSVPLLIQD--LRVSDS------AVYYCALR---- V30.1.2 ---CDCA-ESVDQNTRVETSV-EEGSVSISCTYQTSDPS---PNLFWYQQKPN-TIPKHMMTIITTTVQ-----NDKDFE-EERFGAKHDKTL------KSVPLLIQD--LRVSDS------AVYYCALR---- V30.0.3 ---CDCA-ESVDQNTRVVTFV-EEGSVTINCTYQTSDPS---PYLFWYQQKPN-TIPKHMMTIITTTVQ-----NDKDFE-EERFGAKHDKTL------KSVPLLIQD--LRVSDS------AVYYCALR----

* **** *** V30.0.1 ---CDCA-ESVDQNTRVETAV-EGGSVTINCTYQTSDPS---PYLFWYQQKPN-TIPKYMMMIFATTVQ-----NDKDFE-EERFSAKHDKTL------KSVPLLIQD--LRVSDS------AVYYCALR---- V33.0.10 ---CRGQ-DRVEQVEGEMSDS-EGNHVIISCNY-STSQT---AYLFWYKQLPN-RSPTFILSQY----------SAEADF-TKRFSATLDSKE------KSFPLTIKN--LQVSDS------AVYYCALR---- V33.0.11 ---CRGQ-DRVEQAEGEMTES-ERNHVIISCNYITTSTT---VYLFWYKQLPN-RSPTYILSQY----------SAEADF-KERFSATLDSKE------KSFPLTIKN--LRVSDS------AVYYCALR---- V33.0.13 ---CRGQ-DRVEQPIREMTES-IGDKVVISCIYSTTYTS---LTLFWYKKLPN-RSPTYILNQY----------STEADF-KERFSATLDSTE------KSFPLTIKN--LHVSDS------AVYYCALR---- V33.0.9 ---CRGQ-DRVEQPSIEMTES-EVEEVIIRCNYSTLSTS---PDLFWYKQLPN-RSPTYILNQY----------STEADF-KERFSATLDSKE------KSFPLTIKN--LRVSDS------AVYYCALK---- V33.0.12 ---CRGQ-DKVEQPIREITEG-EGAHVFVQCNY--TSSN---PTLFWYKQLPN-RSPTLILSQY----------SAEAD--KKRFSATLNSKE------KSFPLLIQD--LQVSDS------AVYYCALE---- V33.0.6 ---CRGQ-DKVQQPSGEKTEN-EGDQVNLMCNYTTTASDT-NIYLFWYKQLPN-RSPTFILNKSPFSEG-----TIEPEF-NKRLSATLNSTS------KTVRLLIQD--LRVSDS------AVFYCALR---- V33.0.7 ---CRGQ-DKVQQPSGEMTEN-EGDQVSLMCNYTTTASDG-NIFLFWYKQLSN-RSPTFILNKIPFSEG-----TIEPEF-NKRFTAKLNSTL------RTVPLLIQD--LRVSDS------AVYYCALK---- V33.0.2 ---CRAQ-DRVEQPTRKKTEN-EGDQITLMCNYTTTSTNA-DVYLFWYKQLPD-RSPTFILNKFPFSEG-----TIEPEF-NKRFSAKLNSTL------RTVPLLIQD--LRVSDS------AVYYCALR---- V33.0.5 ---CRGQ-DRVEQPLRSISAN-ERGQVIISCSYSTTDIN---IYLFWYKQLPN-RSPTFILNKQTYGEG-----ITAADF-KERFSATLNSTA------KSVPLMIRD--LRVSDS------AVYYCALR---- V33.0.8 ---CRGQ-DRVEQPLRDITAN-EGDQVIIPCNYSTTDTN---PYLFWYKQLPN-RSPTFILNKLTYGEG-----TVEADF-KDRFSATLNSTA------KSVPLMIKD--AQLSDS------AVYYCALR---- V33.0.3 ---CRGSSDNVEQPSGQITAN-EADSVHLQCSYSTTFTN---AYLFWYKQLPN-RSPTFILSEFTIGEG-----TTEDEF-KGRFSATLNSTL------KSVPLLIQD--LRASDS------AVYYCALR---- V33.0.4 ---CRGQ-NRVEQPSREMTAD-KGDTVTLPCNYSVTTTN---AYLFWYKQLPN-RSPTFMLSAFTVGKG-----TTEDEF-KERFNATLDKTL------RTVPLLIQD--LQVSDS------AVYYCALQ---- V33.0.1 ---CGEQ-DSVDQPDRELTSA-EGNEVILLCNYKLMSAAN--AYLYWYKQLPD-TSPAFILSEFSIGKG-----KAEPEF-KDRFSAKLDSKL------ETVPLTIKD--VRVSDS------AVYYCALQ---- V18.0 ---CRCE-DAVRQPTASVSAF-TEETITLSCEYYTTDSM---PALLWYRQTKN-FPPQYILVRHNLGNG-----SNSKDFPNNRYQSRLDSAM------GQVTLTVRN--LLLSDS------SVYFCALR---- V30.1.1 --DVILRTKLIRT--QRLNLLLKVIL*PLTAHIRLQQT-----QLYSGTSRKSKESPNTC*IKSPTQLD------TLRKNSMRDFTQLSAKLQ---------FL*QSR--IYECQT------AMYYCALR---- V12.0.1 ----LYSWRRNS----SSLFI*IGHQWSLHN---FVL*------LQWLLQQ*LSHVVSTIQQLQTTVSVFG**SQADPTS*SSNFWTIC*IK*------GE*PCVSGDLLCCISDS------AVYYCALK----

Supplemental Figure 4: Multiple Sequence Alignment of T cell receptor alpha/delta V sequences from Danio rerio. Figure was constructed using Mega 6.0 software. Clustal W methodology used for alignment. The * represent hallmark sequences.

6 D delta sequences <D SEQUENCE> trdd1 CGTTTTTGCTACAGAGTATTTCATTGTGTACTTGGGAC-CACATTGAGGCAATATGCGACAAGCCCCTACAAAAACTC trdd2 CTGTCATTTAATATAAGTTCAGTTTGTGAACACATT---CACAGTCTTGACCTGACAGCTCATAGCACACTACCACAA trdd3 AACGAAACATTGGATCCTTTTCTTTGTGAGTTCAGACTTCACAAGTTTATTGTGGAGAACAAACTCCCGAGGACATGT trdd4 GGTTTTTGTTTCGAGTTGTGTCAGTGTGGATTGGGGTACCACAGTGATTCTTTTCAGCTTTTGTCTCGTACAAAAACC trdd5 AGATTCTCCAGATGATTTTCTGTCTGTGAATTACTG---CACAACTGCAGAGGGTTTTTGTATTGAAGTTTTTCATTG trdd6 GGTTTTTGTATTGAAGTTTTTCATTGTGTCTGGACTAC-CACAGTGATACATTCATTATTAGGCGCTGTACAAAATCT 2 J delta sequences nonamer spacer heptamer F G x P trdj1 GGATATTGTCTGAGGGTTGAATGAAGTG----------TGCCGACCCACTAACTTTCGGAGCTCCCATCCGTCTCACGGTCAATCCAAGT trdj2 TGTTTTTGTAGGGGTTACGCTAGACGTGCATTTTCAGCTACTGATCCTTTAACATTCGGCAAACCGATCACCCTCACGGTAATACCAAGT 111 J alpha sequences nonamer spacer heptamer F G x G traj1 GTTTTATGTCTGGGAATGAAGCACTGTG--GTCATCCGGTTCGAACTTCAAGCTTTTAATGGGATCTGGAACCAAGTTAAATGTGCAGCAAAGT traj2 GGTTTATGTATGTGTGTGTAGCCATGTG-TGAATACTGGCAGCCTTGGAAAAATAATCTTTGGTCAAGGAACTAAACTTTTCATCCATTCAAGT traj3 AGTATTTGATACAGGTTGTAGTGCTGTG----TGGATGCAAATACTAACAAGATGATTTTTGGCAGTGGAACAAAGCTGTTTGTTGAACCAGGCAAGT traj4 TATTTGTTTCTGCACAAAGCACTGTGTGTCTGGTAGTGGAGCCGCAGGGAAGATTATTTTTGGAAAAGGAGTGAAACTCACAGTAAAAATTCAGGT traj5 GGTATTTGGTATAGGCTGTAGCTCTGTG---TGAATACAAGGAAATTATAAGATTATATTTGGTGGCGGAACAAAACTCATCGTTGAACAAGGCAAGT traj6 GGTTTTTGTTTTGTGCTCTGACATTGTG-TGTCTACTGGAATGGCATCAAAGATCTTGTTTGGAACAGGAACAAAACTGATAATCCAAAGCAGT traj7 GGTTTTAGTTATGTGTTGTCGTGATGTG-TGTCTACTGGAGGAGGTTATAAGTTGATCTTTGGATCAGGAACACGATTGATAATCGACGCAAGT traj8 AGTTATTGTGATGGTAAATTGCATTGTG-TGACTTCATCTGGGCTCTACCAGATCTATTTTGGCTCTGGAACCAAACTCATAGTTGGAACAAGT traj9 TTTTTGTTTAGTGATCTGGTATTGTGTGACTAGTGGAGCTGGAGGATTCAAGGTTATCTTTGGGAAAGGCACACAGTTAATAGTTGACAAAAGT traj10 AGTTTCTGTAGAGACAAAATTTAATGTG-TGACTCAAAGTGGAGGCTTGAAAATCATTTTTGGCTCTGGAACCAAACTGACTGTTGAAAAAAGT traj11 GGATTTTGATGTGTGCTGTGACATTGTG-TGAATACTGGAGGTGCAAGAAAGCTTGTTTTTGGGAAAGGCACAACATTAACAGTTGGTTCAAGT traj12 AGTTTTAGTTATTTGGTTTTATAGTGTG-TGAATAGAGCCGGCATGGAAAAGCTTATATTTGGGAAAGGCACACAATTACTCATTGAAACAAGT traj13 TGTTTTTGTGTAGTGATTTTACTGTGTG-TGACTGGAACTGGAGTAAACAAAGTTATATTTGGGAAAGGCACAAAACTCATCGTAAATACAGGT traj14 TTTTTGTTTGGTGCTTTTGCATTGTGTGACTACTGGAGGTGGAGGATACAAGATTATCTTTGGTACAGGCACAAAACTGATAGTTGAAACAAGT traj15 AGTTTTTGCTGTTTGGTTTTGTAATGTG-TGACTAGAGATGGAACCAATAAGCTTCTTTTTGGCAAAGGTACAAATCTGATTGTTGAAACAAGT traj16 TGTTTTTGAGTGGATGTTTAACTGTGTG-TGACTGGAACTGGACTCCAGAAAGTTATATTTGGGAAAGGCACAAAACTCATAATAAATGCAGGT traj17 TTTTTGTTTGGTGGTCTTGCATTGTGTGACTGGTTCAAGTGGAGGCTACAAGCTTATATTTGGGACCGGTACAAAGTTAATAGTTGAAACAAGT traj18 AGTTTTTGCTGTTTGGTTTTGTAGTGTG-TGACTGGAAGTGGATTCAATAAGATTATATTTGGCAAAGGCACAAATCTGATTATTGAAACAAGT traj19 TGTTTTTGTGTAGCTTTGTAATGCTGTG-TGACTGACACTGGAGGGAGGAAAGTAATATTTGGGAAAGGCACGAGGCTCTTTGTAGATTCAGGT traj20 AGTTAGAGTTAGTGTTTCATACTGTGTGAATGATGGAAGTGTTTATGGAAAGCTGGTCTTTGGCTCTGGCACCAAATTATATATTAATGAAAGT traj21 AGTTTTTGTCGTTTGGTTTAGTAGTGTG-TGAATAGAGGGGGAGTCGATAAGCTCATATTTGGAAAAGGCACAAAACTGCTCGTTGAAGCACGT traj22 TGTTTATGTATAGGTGTTTAACTTTGTG-TGAATCAGGCTGGATTTCAGAAACTAATATTTGGAAAAGGCACAAAGCTCACTGTAAATACAGGT traj23 AGTTTTTGCTTAGTGGTCTTGCATAGTG----TGACTGGTGGAGTGAACAAGATTATCTTTGGGACAGGAACAAAGTTGGTAGTTGAATCAAGT traj24 TGTTTTTGTTTAGCTTTATAACTTTGTG-TGACTGATGTCGGAGTAAGAAAATTTATATTTGGGAAAGGCACAAGACTCTTTGTAAATTCAGGT traj25 TTAAAGTTATTGTGTTTTGTGCTGTGTGAATGATGGAAGTGGATATGGAAAGCTGATCTTTGGTTCTGGCACCAAATTATATATTAATAAAAGT traj26 AGTTTTTGCTGTTTGGTTTAGTAGTGTG-CGAATGCAGGAGGACTCAGTAAGCTTATGTTTGGGAAAGGCACAAAGTTGCTCATTGAAACAAGT traj27 TGTTTTTGTGTGCCTATTTTTCTGTGTG-TGACTGGAACTGGAGTACGTAAAATTATATTTGGCAAAGGCACAAAACTCACAGTAAATACAGGT traj28 TGTTTTTGTATAGTAATATTACTTAGTG-TGACTCAGGGAGGAGCTCAGAAAATAATATTTGGGAAAGGCACAAAGCTCACTGTAATTACAGGT traj29 TTTTTGTTTGGTAATCTTACATTGTGTGACTTATGGAGGTGGAGGTACCAAACTAATCTTTGGGACAGGCACAATGTTGGTAGTTAAAACAAGT traj30 TTAGAGTTATGATGTTTCGTACTGTGTGATTGATGGAAGTGCATTTGGAAAGCTGATCTTTGGTTCTGGCACAAAATTATATGTTAATGAAAGT traj31 AGTTTTTGCTGTTAGGTTAAGTAGTGTG-TGAATACAGGAGGACTCAATAAGCTTATATTTGGGAAAGGAACAAAGTTGCTCATTGAAACAAGT traj32 AGTTTTTGATTGATAGTTTTGCAATGTG-------GCACTGGATTCAACAAGGTTATCTTTGGAACAGGCACAAAGTTGATAGTTGAAACAAGT traj33 TGTTTTTGTGTGACTGTAAAACTGTGTG-TGACTCAGACGGGATTACAAAAAATTCTATTCGGCAAAGGCACAAAGCTCACTGTAAATTCAGGT traj34 TTTTTGACTAGTACTCTTGCATTGTGTGAATTATGGAAGTGGAAACTACAAGCTTATCTTTGGGACAGGCACAAAGCTGATAGTTGAAACAAGT traj35 CTAGTTTTTGCTGTTTGCTTTTGTAGTG--TGTGACTGGAGGTTTTGACAAGCTTATATTTGGGAAAGGCACAAATCTGATTATTGAAACACGT traj36 TGTTTTTGTTTAGCTTTATAACTGTGTG-TGACTAACACTGGAGCAGGGAAACTCATATTCGGGAGTGGCACACAGCTTTTTGTAAATTCAGGT traj37 GGTTTTAGTGTTGTAGATTTATGCTGTG----TGACTGGAAGTGGATATAAGATTATCTTTGGGACGGGTACAAACCTTATAATCGAAACTAGT traj38 GGTTATTGGTTTGATGAAATGCAATGTG-TGACTTCATCTGGTCTAAGGCAGATCTATTTTGGCTTAGGAACCAAACTCGTTATTGAATCAAGT traj39 TTTTCGATTAGTGGTCTGACATTGTGTGACTTCTGGAAGTGGTGCTTACAAGCTTTTCTTTGGAACAGGCACAAAGTTGATAGTTGAAACAAGT traj40 TTTTTGCTATGTGGTCCTACATTGTGTGACATCTGGAAGTGGACTTTACAAGGTTATTTTTGGGAAAGGCACAAAGTTGTTTGTTGAAACAAGT traj41 AGTTAGTGTAGAGATGAAATATAGTGTG-TGACTCAGTCTGGAAGCTGGAAAATCCATTTTGGCTCTGGAACCAAACTGACCGTTGAAAGCAGT traj42 AGATTTTGCTGTGTTGTTTCATACTGTG-------TGAATGCTGGACAAAAGCTTATTTTTGGAAGAGGCACAAAGCTAGAAATTCAAACAAGT traj43 TTTTTGCTGTGTGGCTAAATGCTGTGTGAATAGTGGAGTTAATATCAACAAAATATTCTTCGGGACAGGAACAAAATTGATCATCAGAACAAGT traj44 TGTTTTTGTACTGCTGTATAATCCTGTG-TGACTAACACTGGAGGATTTAAAATGATTTTTGGCTCTGGAACAAAACTCACAGTTCTGTCAGGT traj45 ATAGTTACAGTTATGAGGTTTAACAGTG--TGTGGATGCTGGATTTGGAAAGCTCATCTTTGGATCTGGCACAAAATTGCATGTTTATTCGAGT traj46 TGTTTTTGTGCATCGATTTAACTATGTG-TGACTACCAGTGGAGGCGTTAAAATTATATTTGGGACTGGAACACAACTTACAGTACTGTCAGGT traj47 AGTAAATGTTTTGGAGTTCCGTGCTGTG-TGTCTAGTAGCTATGGAGGGAAACTCATTTTCGGTCAAGGAACAAATCTGTTTGTTGACAAAAGT traj48 TTCTCGTTATCAGGTTTCACATTGTGTGAATGCTGGAAGTTCATATGGAAAGCTCATCTTTGGGTCCGGCACAAAATTAGAAGTACACACAAGT traj49 TTCTAGTTATCAGATTTAACACTGTGTGACTACTGGAAGTGGATATGGAAAGATAATCTTTGGTTTTGGCACAAAATTAGAAGTTAATACAAGT traj50 AGTAATTGTTGAGGGAAAATGCGGTGTG-TGACTGGATCTGGAAACTGGAAAATCATTTTTGGCTCTGGAACCAGACTTACTGTTGAAACAAGT traj51 CTTTACTGTCATGGTTGCTAACACTGTG-------TGTCATATTCTGAAAAGATCATCTTTGCACAGGGAACAAAGGTTACCATAAATTCAAGT traj52 ATTTTGCTATGCAGTCTAATAATGTGTGACGTATGGAAGTGGAGTTAATAAAATCATCTTTGGATCAGGCACAAAATTAATCATTGAAACAAGT traj53 TGTTTTAGTGTTGTCATGTATCTGTGTG-TGACTGATAGCGGAGGATGGAAAGTAATATTTGGAAAAGGAACCAAACTGACCATCGAGTCAGGT traj54 GGTAATGTATATGGTTGCTAACACTGTG-------TATCATATTCTGATAAGATGTACTTTGGTGAAGGATCAAGGCTTATTATAAATTCAAGT traj55 GTTTTTTGTTGTGAGATTTTTCATTGTG-TGAAAACTGGTGGATACGGAAAGATAATTTTTGGAGCTGGAACTAAATTAATAGTTGCAGCACGT traj56 TAAGTTTATGTAAAGCCACGTAGCTGTG--TGTGACTAACAACGTGGGACGTATGATATTTGGAAAAGGAACAAAACTTATTGTTGATTCAGGT traj57 GATTTTTGAAATGATGTTTCCTGTTGTG----TGAATGTGGGATTGGAAAAGATCATTTTTGGGAAAGGCACACAATTACTAGTTGAATCAAGT traj58 AGATATTGCTTAGAATTTAACTATTGTG-------TGACACGTGCTGACAAAATTGTATTTGGTCAAGGCACAAAGCTTAATGTCGATTACAGT traj59 TGTTTTCGTCATGATTGTTTGTGTTGTG----TGGATGGTTCTGGAAGAAAGATCATCTTTGGTCAAGGGACAGCTTTAACAGTTCAGTCAAGT traj60 TGTTTTTGTAAAGCCACATAACTCTGTG-TGACTATGACTACAATGGGACGTATGATATTTGGAAAAGGAACAAAACTTACTGTCCATTCAGGT traj61 TGTTTTTGCTGTGAGGTCAGTTAGTGTG----TGAATGATGGGACCTGGAAGCTTCATTTTGGAAAAGGGATGACTTTATATGTGCAGTCAAGT traj62 TGTTTTTGTCATGAGTGTTTGTGTTGTG----TGGATGGTTCTGGGCTGAAAATCATTTTTGGTCAAGGAACAACTTTAATAGTTGAATCAAGT traj63 GCTTTTGGAAGAGGTGCTTGTTACTGTG----TGAATGCTGCAACACAAAAGATCGTCTTTGGATCTGGAACAAAAGTATTCATAGAAACAAGT

traj64 GTTTTTTGTCATGATTGTTTCTATTGTG-TGGATGCTGGTGGAGGGAGAAAGATCATTTTTGGTCAAGGAACAGCCTTAAAAGTTGAATCAAGT traj65 ACATTTTGTCTGAGTGTTTGTTACTGTG----TGAATTATGGGAACAACAAGATCACTTTTGGAAGTGGAACAAAACTGACAGTTGAATCTCGT traj66 TTTTTGTGAAGGCTGCTTAGATTGTGTG------ACTTTGAATGCAAACAAGATCATCTTTGGAGAAGGAACAATGCTACATGTAAAAGCAAGT traj67 TGTAAAAATTGTGGTGTTCAACACAGTG----------TGAATTACAATAAGATCACTTTCGGTCAGGGAACACAGCTTTATGTTTACTCCAGT traj68 AGTTTTTGCTCAGGCTTTTGTTAGTGTG----------TGAATTACGACAAGATCTTTTTTGGAGCTGGAACCAAACTAATCATGGAATCTCGT traj69 AGTTTTTGAAGTTAACCAGATCGCTGTG-------TGACAACTGGAGTAAAGATAATATTTGGAAGGGGTACTCGACTTATTGTGGAGTCAAGT traj70 TGTATAAGTAATTGTAATGTAGTTTGTG-TCTGTGTGACTGCTGGTAACAAGATAATCTTTGGAAAAGGAACACAAGTTCATGTTGAAACAAGT traj71 AATGTTTTGGCTGTGAGGTCAGTTAGTG--TGTGAATGATGGGACTAGGAAGATTCATTTTGGAAAAGGGACGACTTTATATGTGCAGTCAAGT traj72 TGTTTTTGTCATGATTGTTTGTGTTGTG----TGGATGGTGCTGCAAGAAAAATCTTTTTCGGTCAAGGAACAACTTTAACTGTTCAATCAAGT traj73 GGTTTTGGAAGTGGTGTTTGTTACTGTG----TGAATGACGCAACACGAAAGATCATCTTTGGGTCTGGAACAAAACTATTCATAGAATCAAGT traj74 AGTTTTTGTCATGATTGTTTGTGTTGTG-------TGGATGCTGCTAGGAAGATCATTTTTGGTCAAGGAACAACTTTAATAGTTGAATCAAGT traj75 AGTTTTTGTGAAGGCTGCTTATATTGTG----TGACTTTTGGTGCAACCAAGATCATCTTTGGAGCAGGAACGATGTTATACGTAGAAGCAAGT traj76 AGTTGTTGTTCTGGTGTTCAACACTGTG-------TGAATTACGGCAACAAGATCATATTTGGTCAGGGAACACAGCTTTATGTTTATGCCAGT traj77 AGAGTTTTTGCTCAGGCTTTTGTTAGTG--------TGTGAATTACAACAAGATCACTTTTGGAGCTGGAACCAAACTAATTGTGGAATCTCGT traj78 GGTTTTTGTAGTTATCCAGATCGTTGTG-------TGACATCTGGAGTAAAGCTAATATTTGGAAGTGGTACTAGACTAATTGTGGAGTCAAGT traj79 AGTTTTTGCATTGTAGTTTGTGTCTGTG-------TGACTGCTGGTTACAAGTTAATATTTGGAAAAGGAACACAAGTTCACGTTGAAGCGAGT traj80 AGTTTTTGTGCTGTGGTTAATTGATGTG-------TGTATGTCGGCAATAAGATTGTGTTTGGAAGAGGGACACAATTATATGTCACTGCACGT traj81 GGTATTTGTGTAGAAGTCATTAGTTGTG-------TGAATGACGGAGTTAGAATAATATTTGGCAAAGGAACAGAACTCAAAATACAACAAAGT traj82 ATTTTGCTTGCACGGTTTTCATAGCGTG---------ACATCAAACTGGAAAGTATTTTTTGGTTCAGGAGTTAAACTACAAGTTGAGCAAAGT traj83 AGTTTTTGGTGCATGTCTAAAGACTGTG-------TGAATACTGGCTATAAGATTATCTTTGGCACTGGAACCAAACTTATCATTCAAACAAGT traj84 TGTTTATGTTATGGATTCAGCTTATGTG-------TGACTAATGCTCAAAAGATAATCTTTGGGACGGGGACAACACTCACTGTACAAACACGT traj85 GGTTTTGAGTTAATGGTTAATGACTGTG-------TGAATACTGGATATAAGATGGTTTTTGGAAAAGGGACCAAACTTGTCGTACAAACGAGT traj86 TGTTTTCGTTATGGATTCAGCAAATGTG-------TGACTAACAACCGAAAGATAGTCTTTGGGACGGGCACATCACTCACTGTACACACACGT traj87 TGTTTTTGTAATGGATTCAGCATATGTG-------TGACTAACAACCAAAAGCTAATCTTTGGGACGGGGACAACACTCACTGTACATACCCGT traj88 TGAGGTTGAGATGAAGTCAGTATTTGTG-------TGAATGCTGCCAACAAGATTATATTTGGGAGTGGGACAAGATTAATAACAGAAACAAGT traj89 TGTTTTTGCACAGACAAAGTTCATAGTG----TGACATCCTCACAATGGAAATTAGTGTTTGGGGAAGGAACTAAATTAACCGTTGAACCAAGT traj90 GTAGTTTTTGGCACATGCTCAAAGGGTG--------TGTGAATGTCAATAAGATTACTTTTGGCAAAGGAACCAGACTGATTGTGCGAACAAGT traj91 TTAATGTAGTGTACTGTACTGCTGTGTG---------AATAATAACTACAAGATCATATTTGGAAGCGGCACCAGTCTCTTTGTTTCCTCAAGT traj92 TTTTTGGTATGTGACCAATCGCTGTGTG---------AATGATGCGTATAAGATTTACTTTGGTAAAGGGACCAAAGTCATAATACAGTCTAGT traj93 TAAATGTACCATGCTGTACTAATGTGTG------AAAGCTGGATACAAGAAAATCATATTTGGACAAGGCACTAAACTAACAGTTGATTCACGT traj94 TGTTGCTAGTTTTTAGTATGTGGCTGTG-------TGACTGATGGACTTAAACTTTTCTTTGGCACTGGGACGAAACTTCTCGTACAAATTAGT traj95 TTTATGTGGTCCAGTAAACCACTGTGTG------ACTCAAGGGGTTAACAGAATTATTTTTGGAAAAGGCACTCAACTCACTGTGCTTTCAAGT traj96 TTTTTAGTACGTAATGAATGACTGTGTG---------AATGATCGAATTAAACTGCTATTTAGCAAAGGAACAAAACTCATTGTACAGTCAAGT traj97 TAAATGTACTGTGCTTTACTGATGTGTG------AATCCTGGAGGAACCAAGCTCATATTTGGCTCAGGCACCAAACTAACAGTTGTTTCAAGT traj98 ATATGAGTATGCGATCAACAGCAGTGTG------ACTCAAAGTGGATTCAAATTTATATTTGGAAAAGGCACTAAACTAACCGTGCTTTCCAGT traj99 AGTTTTTGGCATATAGTTAAAGACTGTG-------TGAATGCTGGACTTCAACTTTTCTTTGGAACAGGGACAAAACTTGTGGTACAAATCAGT traj100 AGTTATTATCCCTCTACAAACCACTGTG----TGAATAACAATTTTGGTAAATTAATATTTGCGAGTGGGACACAACTGTATGTTCAACCAAGT traj101 TTTCTGTAATGAGATTTATAGTTGTGTG------ACTCAAAATGCATTCAAACTGATATTTGGAGAGGGGACGCAGCTCTTTGTGTTATCCAGT traj102 AGTATTTATAACACAGACAGTCACTGTG----TGAATGACAACAATTACAAAGTGATATTTGCGAACGGAACTCAGTTGTTTGTACAAACAAGT traj103 AGTTTTTGGTATGTGGCTAAAGACTGTG-------TGAATGACGGACGTAGACTTTTCTTTGGTGCTGGGACAAAACTTCTTGTACAAATCAGT traj104 AGTTTTTATTCCACCACTAACCACTGTG----TGAATGGCAATACTGGTAAAATAATATTTACGAATGGCACCCGACTATATGTACAAACAAGT traj105 TGTTTCTGTGCTGAAGTGTATAACTGTG----TGAATCAAAACACAAACAAACTAATATTTGGACAGGGGTCGCAGCTTGTTGTGTTATCCAGT traj106 AGTATTTATAACACAGACAGTCACTGTG----TGAATGACAACTACGGCAAAATGATATTTGCGAAGGGAACTCGGTTGTTTGTACAGACGAGT traj107 GGTTTTTGTGCTGATAGATCAGCCTGTG-------GGAATAATAATATCAAAATTTTCTTTGGAAGAGGAACAAAATTAACAGTACAATCAAGT traj108 AGTTTTTCGTATTAGGCTGAAGACTGTG-------TGAATGATGGATTTAAGCTTTTCTTTGGCACGGGGACAAAAGTTGTAGTACAAATTAGT traj109 AGTTATTATCCCTCGACTAACCACTGTG----TGAATGACAATATCGGCAAAATTATATTTGCGACTGGGACACAAGTGTATGTACAACCAAGT traj110 GGTTTTTGTACTCGTATATCAATCTGTG-------TGACTACTAGCGTTAAAATTGTTTTTGGAAGAGGAACAAAATTAACAGTGCAATCAAGT traj111 TTATTATATGTGCATTTATCATTGTGTG------ACGAACACTGCTGACAAAATCATATTTGGAAAAGGGACACAACTATTTGTGCACTCAAGT

Supplemental Figure 5: TCR α and δ D and J sequences. Sequences aligned by RSS and conserved FGxG(P) sequences. Spaces were introduced only to align sequences at these conserved motifs. Terminal GT is underlined.

<M E K Q L M L I L I L T P G V M T A D Q I S P N K E A L T V K E C1 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C2 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C3 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C4 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C5 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C6 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C7 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA C8 ATGGAGAAACAACTGATGCTCATTTTAATTCTGACTCCAGGTGTGATGACTGCAGACCAGATTAGCCAAATAAAGAAGCTCTTACTGTAAAGGAA V REGION TCRD V23.2.2 E E T V T F S C S Y D T S S S Y V R L Y W Y R Q Y L N G E P Q C1 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C2 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C3 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C4 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C5 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C6 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C7 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA C8 AGGAGACAGTGACCTTCAGTTGCCTCATATGATACAAGCAGCAGTTATGTTAGGCTTTACTGGTACAGACAATATCTTAATGGAGAACCTCAGTA Y L L F K A A R S S S G G G R P D N P R F K S T T S D S S T E L C1 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C2 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C3 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C4 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C5 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C6 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C7 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA C8 TTTATTATTCAAAGCTGCACGATCAAGTAGTGGAGGTGGGAGACCCGATAATCCTCGTTTTAAGTCGACTACATCAGACTCATCCACTGAACTCA

> <D4>< D6 >< J2 T I S G V T L S D S A L Y Y C A L R V G E Y D Y A T D P L T F G C1 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C2 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C3 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C4 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C5 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C6 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C7 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC C8 CTATTAGCGGTGTAACTCTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACGACTACGCTACTGATCCTTTAACATTCGGC >< C REGION K P I T L T V I P K E T V N S P P A F L S V L S P I K G H G C1 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCA-TGGA-T C2 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCA-TGGA-T C3 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCA-TGGA-T C4 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCCGTCTTGTCCCCTATAAAGGGCCAATGGAGT C5 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCAATGGA-T C6 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCAATGGA-T C7 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCA-TGGA-T C8 AAACCGATCACCCTCACGGTAATACCAAAAGAGACAGTGAATTCTCCTCCGGCATTTTTGTCTGTCTTGTCCCCTATAAAGGGCCA-TGGA-T S D I C V A A G F F P C1 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C2 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C3 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C4 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C5 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C6 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C7 CTGATATTTGTGTGGCCGCCGGATTCTTTCC C8 CTGATATTTGTGTGGCCGCCGGATTCTTTCC Supplemental Figure 6: Sequences of clones Sanger sequenced.

V sequence 23.2.2 > <D4>< D6 >< J2 ><Cδ L S D S A L Y Y C A L R V G -E Y _ D Y A T D P L T F G K P I T L T V I P K E 1 CTGTCAGATTCAGCTCTCTATTATTGTGCTCTAAGAGTAGGAGAGTACG-ACTACGCTACTGATCCTTTAA-CATTCGC-AAA-CCGATCACCCTCACGGTAA-TACCAAAA-GA ----Cδ---- T V N S P P A F L S V L S P I K G H G S I D C V A A G F F P S I T I C 1 GACAGTGAATTCTCCTCCGGCAT-TTTTGTCTGTCTTGTCCCCTATAAAGGGCCATGGATCTGATATTTGTGTGGCCGCCGGATTCTTTCCGATCTCTACTATATGC Supplemental Figure 7: Sequence of clone obtained from PacBio sequencing. A total of 440 sequences having identical CDR3s were found. Sequence presented has been trimmed to show just the terminal portion of the V segment and the initial portion of the C segment to highlight the CDR3.

Supplemental Table 1. Primers used in PCR

Primer Name For/Rev Domain Sequence Priming Site

MFC525 R TCR δ C region 5’-GGAAAGAATCCGGCGGCCACAC-3’ VAAGFF

MFC526 R TCR δ C region 5’-TGCTTGACAAGGACAGGACTGCA-3’ AVLSLSS

MFC527 R TCR δ C region 5’- TGCCTTTGCTGTTTTTCCATCCA-3’ DGKTAKA

RACE 5’ F 5’-CGACTGGAGCACGAGGACACTGA-3’

RACE 5’ NESTED F 5’-GGACACTGACATGGACTGAAGGAGTA -3’

MFC535 F TCR δ V region 5’-TACTGGTACCGACAG-3’ YWYRQ

MFC536 F TCR δ V region 5’-TAYGGTAYMGNCAR -3’ YWYRQ

MFC537 F TCR δ V region 5’-CTCTAYTGGTAYMGNCARTAT-3’ LYWYRQY

MFC561 F TCR α C region 5’-CTCATGCCTGGCAACTGACTTCAC-3’ SCLATDFT

MFC562 R TCR α C region 5’-TCAGCCAGAAGATGCCCAGTGACA -3’ SLGIFWL

MFC565 F TCR β C region 5’-CCACATAGCCATACAGGACAAGAC -3’ PHSHTGQD

MFC566 R TCR β C region 5’-CAGGATGTAGCCAAAGCCAACCAGC -3’ QNAKAVDQ

MFC559 F TCR δ C region 5’-CAGTCCTGTCCTTGTCAAGCA-3’ AVLSLSS

MFC560 R TCR δ C region 5’-GTGTGACATTCAAGTGTAGCCG-3’ LLAKCVCV

MFC563 F TCR γ C region 5’-CCTGGGAAGGACAGTGTTGTGAc-3’ PGKDSVVT

MFC564

MFC534

R

TCR γ C region

Barcoded RACE 5’ NESTED

5’-GTGGCTGTGCTCCACAATCTGG-3’

5’-TCAGACGATGCGTCATGGACACTGACATGGACTGAAGGAGTA-3’

QIVEHSH

MFC579

Barcoded TCR δ C

region

5’- GCATATAGTAGAGATCGGAAAGAATCCGGCGGCCACAC-3’

Barcode/CVAAGFFP

Genomic organization of the zebrafish (Danio rerio)Tcell ...vetmed.tamu.edu/media/1777265/seelye imgt 2016 zebrafish tcr delta.pdfthe TCRα/δ locus of Danio rerio based on the most

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