High-Throughput Single-Cell Sequencing of Both TCR-Β Alleles

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High-Throughput Single-Cell Sequencing of both TCR-β Alleles Tomonori Hosoya, Hongyang Li, Chia-Jui Ku, Qingqing Wu, Yuanfang Guan and James Douglas Engel This information is current as of September 29, 2021. J Immunol published online 31 October 2018 http://www.jimmunol.org/content/early/2018/10/30/jimmun ol.1800774 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 31, 2018, doi:10.4049/jimmunol.1800774 The Journal of Immunology

High-Throughput Single-Cell Sequencing of both TCR-b Alleles

Tomonori Hosoya,*,1 Hongyang Li,†,1 Chia-Jui Ku,* Qingqing Wu,* Yuanfang Guan,† and James Douglas Engel*

Allelic exclusion is a vital mechanism for the generation of monospeciﬁcity to foreign Ags in B and T lymphocytes. In this study, we developed a high-throughput barcoded method to simultaneously analyze the VDJ recombination status of both mouse TCR-b alleles in hundreds of single cells using next-generation sequencing. The Journal of Immunology, 2018, 201: 000–000.

ertebrates have evolved both innate and adaptive immune that of the functional allele (7, 8), traditional RNA-based methods systems to protect individuals against infection, cancer, cannot detect the mechanisms underlying allelic exclusion. One

V and invasion by parasites. B and T lymphocytes com- major challenge in analyzing the genes themselves is that there is Downloaded from prise the central components of adaptive immunity, and every only one copy of DNA representing each allele, unlike the exis- individual lymphocyte harbors specific reactivity to a single Ag that tence of multiple transcribed RNA species. Therefore, extremely is conferred by individually unique Ag receptors expressed on the accurate DNA sequencing is required to avoid erroneously mis- cell surface. The diversity of Ag receptors is generated by DNA assigning nucleotide-level mutations that would alter the interpre- recombination, called VDJ rearrangement in jawed vertebrates (1). tation of TCR locus activity. Additionally, no reference genome is To maintain the required monospecificity of mature lymphocytes, available for merging multiple sequencing reads due to the many http://www.jimmunol.org/ only one of the two autosomal alleles is allowed to express millions of possible sequences that can be generated from VDJ functionally rearranged b- and a-chain TCRs (TCR-b and TCRa) rearrangement (9). Thus, de novo sequence assembly or a long-read in T cells or Ig H and L chain receptors (IgH and IgL) in B cells; approach is required to retrieve the original genomic sequence of both are controlled by a historically opaque mechanism referred to both alleles in single cells. To significantly improve the traditional as “allelic exclusion.” Allelic exclusion occurs at the genetic level Sanger sequencing method employed by us and others (10–12), we for IgH, IgL, and TCR-b (2), whereas it occurs at the level of developed a high-throughput method that enables analysis of protein localization on the cell surface for TCRa (3). Loss of Trb allelic exclusion status by sequencing both alleles of the allelic exclusion results in dual TCR expression, which can lead to genome in single cells, enabling us to determine whether each

autoimmunity (4, 5). allele in those cells underwent either no, unproductive, or pro- by guest on September 29, 2021 A high-throughput method to study the diversity of lymphocytes ductive rearrangement. at the single-cell level has been reported recently (6); yet, how one might analyze their monospecificity remains unclear. Because the Materials and Methods abundance of transcripts from a nonfunctional allele is lower than Single cell isolation Staged thymocytes were first isolated from mice (C57BL/6J; The Jackson *Department of Cell and Developmental Biology, University of Michigan, Ann Ar- Laboratory) between 5 and 8 wk old using a cell sorter (BD FACSAria III). † bor, MI 48109; and Department of Computational Medicine and Bioinformatics, Lin2CD42CD8a2 Thy1.2+cKit2CD25+CD282 (double negative [DN] 3a University of Michigan, Ann Arbor, MI 48109 stage), Lin2CD32TCR-b2 CD8a2Thy1.2+cKit2CD252 (DN4 stage), 1T.H. and H.L. contributed equally to this work. CD4+CD8+ (double positive [DP] stage), and TCR-b+CD4+CD8+ (late DP ORCID: 0000-0001-9593-1825 (J.D.E.). stage) thymocytes (10,000 to 100,000 cells at each stage) were isolated as described previously (10). Next, single cells were directly sorted into 20 ml Received for publication June 22, 2018. Accepted for publication October 1, 2018. of lysis buffer (containing 13 Q5 Reaction Buffer [New England Biolabs], This work was supported by National Institutes of Health Grant AI094642 (to T.H. 4 mg Proteinase K, and 0.1% Triton 3100) in one well of a 96-well PCR plate and J.D.E.). The research was also supported in part by a University of Michigan using a Synergy cell sorter (Sony iCyt SY3200). The cell sorter setting was Comprehensive Cancer Center Support grant (P30 CA046592) for use of the Flow carefully aligned so that sorted cells were precisely deposited into the center of Cytometry and DNA Sequencing Cores at the University of Michigan. each well. Sorted single cells in the lysis solution were kept on ice and then T.H. designed the study, performed experiments, wrote computer script, analyzed the digested at 55˚C for 60 min followed by further digestion at 95˚C for 15 min data, and wrote the manuscript. H.L. designed the study, wrote computer script, and (toinactivateProteinaseK)usingaPCRthermalcyclerwithin6haftersorting. edited the paper. C.-J.K. designed the study, performed experiments, analyzed the data, and edited the manuscript. Q.W. performed experiments and edited the paper. Multiplex nested PCR Y.G. designed the study and edited the paper. J.D.E. designed the study and edited the paper. For the first round of PCR, the following primers were selected to amplify Address correspondence and reprint requests to James Douglas Engel, University of all potential VDJ rearrangement at the Trb locus: 31 V region primers Michigan Medical School, 3035 Biomedical Science Research Building, 109 Zina covering all 35 Trbv genes (13), two D region primers, two J region pri- Pitcher Place, Ann Arbor, MI 48109. E-mail address: [email protected] mers, and two control primers used to detect sequences 39 of the Actb gene The online version of this article contains supplemental material. (Fig. 1A, 1B). The full list of primers used are deposited at (https://umich. box.com/s/3f5q64u2i68dn2i7hlneucxph9oetpvb). Because this method Abbreviations used in this article: CCS, circular consensus sequencing; DJ, recom- was designed to recover not only rearranged but also germline-configured bined diversity and joining segments; DN, double negative; DP, double positive; 9 GL, germ line; PacBio, Pacific Biosciences; pVDJ, productively recombined VDJ genomic DNA, the two J primers were designed to be 3 of J1–7 and J2–7. Vb8 b segments; Tg , Vb8 transgenic mice; uVDJ, unproductively recombined VDJ The following PCR condition amplifies the entire J1 (J 1 primer coupled segments. with a V or D region primer) as well as J2 genomic DNA region (Jb2 primer coupled with a V or D segment primer). Because VDJ will be Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 spliced to a C region at the RNA level, the selected Jb primer sequences

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800774 2 VALIDATED SEQUENCING OF BOTH Trb ALLELES IN SINGLE CELLS remain in genomic DNA after recombination. D region primers were agarose gels with 0.4 mg of 2-Log DNA Ladder (New England Biolabs) designed 59 of D1 and D2. The D segment primers amplify only a D-to-J (Fig. 1C). Eight samples were selected from each 96-well PCR plate. Of rearranged genome, but not a V-to-DJ rearranged genome. After V-to-D1-to-J note, the PCR conditions employed in this study allow amplification of rearrangement, the D1 primer sequence is removed from the genome. unrearranged (germline configuration) genomic DNA (Fig. 1C left panel). Similarly, after V-to-D2-to-J2 rearrangement, the D2 primer sequence is removed from the genome. The first round of PCR was performed in a Recovery frequency m 3 60- l final reaction volume containing of 50 nM of each primer, 1 Q5 To analyze the frequency of recovered wells, a primer pair amplifying the 39 Reaction Buffer, 200 mM each deoxynucleotide triphosphates, 0.4 U Q5 region of the Actb gene was added to the first round PCR. One microliter of High-Fidelity DNA Polymerase (New England Biolabs), and 20 ml of the the first round PCR product was used in a PCR reaction with the following lysed single-cell solution. The PCR condition was 30 s at 98˚C followed by nested Actb primers: 59-CATAGGCTTCACACCTTCCT-39,59-CTTTGC- 30 cycles of 5 s at 98˚C, 10 s at 66–58˚C, and 2 min at 72˚C and then final CTCCATCTGCATAAC-39, and FAM-labeled probe 59-TGCTAGTCTGA- extension for 2 min at 72˚C. During the first five cycles, the annealing AGCTGCCCTTTCC-39 (ZEN/Iowa Black FQ) purchased from Integrated temperature was reduced 2˚C per cycle from 66 to 58˚C and then per- DNA Technologies. Luna Universal Probe qPCR Master Mix (M3004; New formed at 56˚C for the last 25 cycles. England Biolabs) was used in a 20-ml reaction. The PCR condition was 30 s For the second round of PCR, the following nested primers were selected: 32 at 95˚C followed by 45 cycles of 1 s at 95˚C and 20 s at 60˚C using Ste- nested V region primers containing forward adapter sequences (AF-Vbn), two pOnePlus Real-Time PCR System (Thermo Fisher Scientific). A 90–95% nested D region forward adapter primers (AF-Dbn), and two nested J region recovery efficiency was routinely obtained from each plate containing 96 primers in a reverse adapter orientation (AR3-Jbn). The second round of PCR single cell–sorted wells. was performed in a 20-ml final reaction volume containing 50 nM of each primer (Fig. 1A), 13 Q5 buffer, 200 mM of each deoxynucleotide triphosphate, Pacific Biosciences high-throughput sequencing m 0.4 U Q5 High-Fidelity DNA Polymerase (New England Biolabs), and 1 lof Because the length of the PCR products described above ranged from 200 the first round PCR product. The second PCR condition was same as for the first to 2700 bp (Fig. 1B), a sequencer with the ability to read long DNA Downloaded from round PCR but for only 25 cycles. fragments is preferred. To this end, Pacific Biosciences (PacBio) single Finally, in the third round of PCR, unique barcoded–forward adapter and molecule real-time sequencing technology (14) was employed. A PacBio barcoded–reverse adapter 3 combinations of primers were selected for each smartbell adapter was ligated to the barcoded PCR product mixture and single cell (Fig. 1A). Barcodes were adopted from a published report (6). then sequenced on a PacBio RS II sequencer at the University of Michigan The third PCR condition was same as for the second round PCR (5 + 20 DNA Sequencing Facility. Thus, each circular consensus sequencing cycles). Hot Start PCR was performed for all PCR reactions using either a (CCS) read generated from PacBio sequencing corresponds to a single BioRad T100 or Applied Biosystems 2720 Thermal Cycler. All primers were

molecule of initial PCR product (Fig. 2). http://www.jimmunol.org/ purchased from Integrated DNA Technologies. The 37 (for first round) or 36 (for second round) PCR primers at 200-mM final concentrations were mixed, PacBio raw reads analysis aliquoted, and frozen for subsequent use. Each PCR mix was prepared im- mediately before initiating PCR reactions. (The amplification efficiency was To obtain DNA sequences amplified from genomic DNA in single cells, we reduced if the PCR mix was prepared in advance and repeatedly frozen and developed an in-house analysis pipeline (Fig. 2; the full version is available thawed. Amplification efficiency was also reduced when 500 nM of each at https://github.com/Hongyang449/scVDJ_seq). To calculate CCS reads primer was used in the second round PCR reaction). Successful DNA am- of inserts, PacBio ConsensusTools software version 2.0.0 was used with plification was confirmed by running 2 ml of third round PCR product on the following settings: –minFullPasses = 2, –minPredictedAccuracy = 90, by guest on September 29, 2021

FIGURE 1. High-throughput Trb genomic DNA sequencing from single cells. (A) Strategy for multiplex PCR amplification of genomic DNA sequences at the Trb gene VDJ loci from single cells. Final PCR products were mixed and then sequenced by PacBio RS II sequencer. Details are described in Materials and Methods.(B) Schematic presentation of VDJ rearrangement at the Trb locus with simplified illustration of multiplex primer location. Genomic DNA recombination events are initiated by recombining Db (diversity) and Jb (joining) segments on both chromosomes. Subsequently, on only one chromosome, one of the Vb (variable) segments is joined to the previously rearranged DJ recombinant. Not only the selection of each VDJ segment but also multiple lengths of spacer sequence between V, D, and J generates an incalculable number of VDJ sequence possibilities. mRNA splicing joins Cb (constant) segments to the rearranged VDJ DNA recombinants to generate a final TCR-b protein. VDJ rearrangement that generates a stop codon or elongated transcript results in a predictedly unproductive TCR-b.(C) Successful amplification of rearranged VDJ, DJ, and germline D-to-J regions were confirmed by agarose gel electrophoresis with New England Biolabs 2-Log DNA Ladder in the last (left panel) and first (right panel) lanes. AF, adapter forward; AR, adapter reverse; BC, barcode; Dbn, D region primers; Jbn, J region primers; Vbn, V region primers. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. A schematic outline of the PacBio raw data processing. Details are described in Materials and Methods.

–minLength = 10, and default for other parameters (https://github.com/ Next, we generated subgroups in each single cell (unique barcode set) PacificBiosciences/SMRT-Analysis/wiki/Documentation). based on the presence of a V or D primer sequence, and sequences in each First, to demultiplex CCS reads, we grouped the CCS reads based on the subgroup were aligned using the multiple sequence alignment software presence of a barcode primer set flanking each end of the CCS. We obtained MUSCLE (15) (http://www.drive5.com/muscle/). For each group, hierar- ∼35,000 CCSs per PacBio RS II SMRT Cell on average, and 46% of them chical clustering based on sequence length was performed to create initial started with a barcode-A forward primer and ended with a barcode-A clusters, then k-means clustering was recursively performed on the DNA reverse 3 primer. The remaining 54% had mutations, deletions, or addi- sequences of each initial cluster to generate the final multiple clusters until tions within the barcoded primer sequence or bore truncated barcode every nucleotide position in each cluster achieved .51% identity. Clusters primer sequences and were excluded from the analysis. containing only one CCS read were excluded. Aberrant CCSs with two

FIGURE 3. Trb VDJ rearrangement status in single thymocytes. (A) Cluster tags in a total of 5877 clusters recovered from single wild type thymocytes at DN4, DP, and late DP (post–b-selection) stages. Each cluster consensus sequence was categorized into one tag shown on the x-axis (see Supplemental Fig. 1 for detail). (B) Calculated Trb allele status for 909 single wild type thymocytes at DN4, DP, and late DP (post–b- selection) stages. The data in (B) represent a summary of four mice for DN4, ﬁve mice for DP, and four mice for late DP stages, and details are shown in Supplemental Figs. 2 and 3. 4 VALIDATED SEQUENCING OF BOTH Trb ALLELES IN SINGLE CELLS

PCR products ligated together that probably resulted during the smartbell adapter ligation step were also excluded from the analysis. To obtain accurate genomic DNA sequences, we calculated a consensus sequence from multiple CCSs in each cluster. Of note, each CCS file that was generated from single zero-mode waveguide corresponds to one copy of PCR product. When a mutation happened during the first PCR cycle, 50% of the final PCR products inherit the mutation at a specific base position in the final PCR product. Similarly, a mutation during the second cycle is inherited by 25% of the final PCR products, 12.5% is inherited after the third cycle, and so on. After adapter and J region genomic DNA sequences (except the J region used for rearrangement) were removed, the cluster consensus with V primer sequences were submitted to IMGT/HighV-Quest analysis (16) (http:// www.imgt.org/) to determine whether a specific VDJ sequence in a cluster was predicted to encode a productive or unproductive TCR-b protein. For example, when DNA sequences rearranged with J1–3, J1–4 to J1–7 regions, as well as 39 of J1–7 to J1, primer sequences were removed. The removal of the extra J region genomic DNA is essential to accurately determine the productivity of rearrangement, especially when some bases are truncated from J regions. Based on sequence and functionality predicted by IMGT/HighV- Quest analysis, each cluster consensus sequence was categorized into a tag shown in Supplemental Fig. 1A. Because the status of an allele Downloaded from (germ line [GL]; recombined diversity and joining segments [DJ]; unproductively recombined VDJ segments [uVDJ]; and productively recombined VDJ segments [pVDJ]) can be determined from possible combinations of one to two cluster tags (a total of 24 different patterns are shown in Supplemental Fig. 1B), the rearrangement status from within a single cell (the combination of two separate allelic patterns) was calculated from two to four tags recovered from each cell (for a total 253 different combinations of tags). From those combinations, the http://www.jimmunol.org/ summary tables were generated. In this study, the rearrangement status of both alleles was obtained from 909 single cells (25% of 3664 wells) for thymocytes post–b-selection and 193 DN3a stage thymocytes (25% of 784 wells) when V-to-DJ rearrangement takes place. The frequency depends on sequence depth and was 40% when we recovered 29,643 CCSs from 576 wells. We obtained zero to four clusters per single cell (1.62 clusters per cell on average) in this depth of sequencing. PacBio CCS data, the barcode list for each sample, genomic DNA, and CDR3-IMGT/HighV-Quest amino acid sequences for all clusters generated from this study are deposited at (https://umich.box. by guest on September 29, 2021 com/s/3f5q64u2i68dn2i7hlneucxph9oetpvb).

Results High-throughput Trb genomic DNA sequencing from single cells To sequence both alleles of genomic DNA in the Trb gene VDJ region, we employed a multiplex strategy (Fig. 1A; details described in Materials and Methods) because mouse thymocytes were directly sorted into lysis buffer in individual wells of 96-well PCR plates. Multiplex, nested PCR reactions followed by barcoding PCR, amplified unrearranged configuration genomic DNA and all potential rearranged DNA in the Trb locus (13) (Fig. 1B, 1C). The PCR products were mixed and then sequenced in a PacBio RS II long-read sequencer (www.pacb.com). Each circular consensus sequence (CCS) read of an insert generated by the PacBio system corresponds to a single molecule of the original PCR product (Fig. 2). Based on barcoding and sequence similarity, multiple CCSs were grouped into clusters (Fig. 2). For each cluster, the consensus sequence was retrieved that corresponds to an original PCR product. The consensus sequences were subsequently submitted to IMGT/HighV-Quest analysis (16) (http://www.imgt. org/) to determine whether the VDJ sequences in any cluster predicted that a functional TCR-b protein could be generated in that FIGURE 4. V and J region usage. Frequency of each V region (A) and J cell. From one Trb allele, two [(V-)D1-J1 and (V31-)D2-J2 rear- region (B) use was calculated out of 3356 VDJ clusters recovered from rangement] or one [(V-)D-J2] PCR product is amplified (Fig. 1B, wild type single thymocytes at DN4, DP, and late DP stages. Stage-specific Supplemental Fig. 1). Theoretically, between two and four PCR data are shown in Supplemental Fig. 4. products (clusters) can be recovered from two alleles in each cell. Details for the computational processing of the PacBio output data To calculate the PCR error rate under our experimental con- to calculate allele rearrangement status are described in Materials ditions, we took advantage of the unrearranged (germline and Methods. configuration) genomic DNA corresponding to parts of Trbd1 to The Journal of Immunology 5

FIGURE 5. Endogenous Trb rearranged status in thymocytes of TgVb8. Number of tags (A) for recovered PCR fragments and rearrangement status (B) calculated from late DP stage single cells isolated from wild type (black) and TgVb8 (Tg, white). Data represent the summary from four mice. Downloaded from

Trbj1–7 and Trbd2 to Trbj2–7 (Fig. 1B) in single bone marrow productive and 2158 unproductive) recovered from single thy- cells. From single bone marrow cells we obtained two PCR bands mocytes in this analysis are available online (see https://umich. (Fig. 1C) that correspond to the lengths of germline DNA am- box.com/s/3f5q64u2i68dn2i7hlneucxph9oetpvb). Surprisingly, plified using Db1 forward and Jb1 reverse nested primers (2672 we recovered D2-to-J1 rearranged sequences, although only b b bp) as well as D 2 forward and J 2 reverse nested primers (1896 rarely (Supplemental Fig. 2). At the DN3a stage, when V-to-DJ http://www.jimmunol.org/ bp). The accuracy of the resultant sequences was 99.768% for rearrangement takes place, only 9% of the cells had generated a each individual CCS, including both PCR and PacBio sequencing productive allele (0.5% pVDJ/GL, 7.2% had generated a pVDJ/ errors. To increase the accuracy, we employed an approach to DJ recombinant, and 1.5% were in a uVDJ/pVDJ configuration, obtain the consensus sequence from more than two CCSs. When Supplemental Fig. 3), all of which probably represent cells that we obtain only two CCSs per cluster, an error at a position results were about to develop to the next (DN3b) stage. in 50% identity at the position (e.g., an A in one CCS and a G in Next, to determine whether the methods developed in this study the other CCS); such a cluster was then removed because it is not can capture an artificial allelic exclusion phenomenon, we ana- .51% identical. When we recover three CCSs, an error at any lyzed late DP cells isolated from Vb8 transgenic mice (TgVb8) position results in a 66.6% identity at the position (e.g., an A in (18), which express a productively rearranged TCR-b protein; by guest on September 29, 2021 two CCSs and a G in one CCS). In this case, A is the consensus expression of that transgene has been shown to repress endog- sequence at the position in this cluster. When a consensus was enous Trb VDJ rearrangement (19). Out of 2429 clusters gen- generated from three randomly chosen synonymous CCSs, the erated, 722 clusters bore the DNA sequence of the TgVb8 accuracy increased to 99.991%. These data demonstrate that this transgene (Supplemental Fig. 4A) and were removed from fur- method using PCR with Q5 High-Fidelity DNA Polymerase (New ther analysis. We then determined the rearrangement status England Biolabs) followed by calculating the consensus sequence of both alleles from 127 single cells (Supplemental Fig. 4B, 4C). from multiple CCSs yields .99.99% accuracy, even after a total V-to-DJ rearrangement was not observed at the endogenous lo- of 80 PCR cycles. cus in 80% of the thymocytes, in agreement with published observations (19). However, 20% of the cells had either Trb VDJ rearrangement status in single thymocytes unproductively (13% uVDJ/DJ) or productively (6% pVDJ/DJ) To precisely determine the genomic status at the Trb loci following escaped transgene-suppressed allelic exclusion to allow V-to-DJ VDJ rearrangement in single cells, we analyzed wild type DN4, DP, rearrangement on one of the endogenous Trb loci. In the TgVb8, and late DP stage thymocytes in which Trb VDJ rearrangement had D2-to-J2 rearrangement was observed less often than D1-to-J1. already been completed (i.e., post–b-selection). Because DJ joining This supports the idea that D2-to-J2 rearrangement was more takes place before the DN3 stage on both alleles, all of the cells frequently repressed when compared with D1-to-J1 region re- analyzed in this study would be predicted to have undergone at least combination in the presence of a functional TCR-b transgene, in one productive VDJ recombination event on one allele. We obtained agreement with published analysis of nine T cell clones (19). a total of 5877 clusters and assessed the rearrangement status of both alleles from 909 single cells (Fig. 3). Fifty two percent (472) of the Discussion single thymocytes were in a pVDJ/DJ configuration and 41% (377) In summary, the method developed in this study provides an were in a uVDJ/pVDJ configuration as expected from the 60/40 rule extremely accurate, rapid high-throughput approach for the (2). Four percent (37) of the post–b-selection thymocytes were in a analysis of Trb genomic DNA status at the single-cell level. pVDJ/pVDJ arrangement, which was similar to the 3% dual TCR- This strategy allowed us to analyze the rearrangement status in b–bearing cells as analyzed using cell surface Abs (17). Stage- thousands of single cells, which is more than 10-fold greater specific data are shown in Supplemental Figs. 2 and 3. The V than the number than from Sanger sequencing methods previ- regions that were most frequently used for recombination were ously employed by us and others (10–12) and requiring far less V13–2 (Figs. 4, 5, Supplemental Fig. 4), which is also the most time. This strategy would also be directly applicable to the frequently used in inflamed lung epitope–specific CD8+ Tcells analysis of the Igl and Igh genes (encoding Igs in B cells), (8). Six thousand four hundred and fifteen V-to-DJ rearranged which are also regulated by recombination and allelic exclusion genomic DNA and CDR 3 (CDR3) amino acid sequences (4258 at the genetic level (2). 6 VALIDATED SEQUENCING OF BOTH Trb ALLELES IN SINGLE CELLS

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