ENHANCED IMMUNE CELL RECEPTOR SEQUENCING METHODS

Description

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/481,936, filed Jul. 30, 2019, which is a national stage filing under 35 U.S.C. 371 of International Patent Application Serial No. PCT/US2018/015819, filed Jan. 30, 2018, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 62/452,409, filed Jan. 31, 2017, the entire contents of each of which are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 5, 2022, is named L046170192US02-SEQ-JRV, and is 150,607 bytes in size.

FIELD

The disclosure relates to methods, systems and kits for sequencing immune cell receptor repertoires from immune cells, such as T-cells or B-cells.

BACKGROUND

Immune cell repertoires, such as B- or T-cell repertoires, consists of millions of lymphocytes, each expressing a different protein complex that enables specific recognition of a single antigen. CD4 and CD8 positive T-cells express so-called T-cell receptors (TCRs). These heterodimeric receptors recognize antigen-derived peptides displayed by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells, as described in Rudolph M G, Stanfield R L, Wilson I A. How TCRs bind MHCs, peptides, and coreceptors. Annu Rev Immunol. 2006; 24:419-66. TCRs are composed of two subunits, most commonly of one α and one β chain. A less common type of TCR contains one γ and one δ chain.

Alpha (α) chains consists of a (variable) V, a joining (J) and a constant (C) region, while beta (β) chains contain an additional diversity (D) region between the V and the J region (see FIG. 1), as described in Starr T K, Jameson S C, Hogquist K A. Positive and negative selection of T cells. Annu Rev Immunol. 2003; 21:139-76. Each of these TCR regions is encoded in several pieces, so-called gene segments, which are spatially segregated in the germline. In humans, the TCR α gene locus contains 54 different V gene segments, and 61 J gene segments. The human TCR β chain locus comprises 65 V, 2 D and 14 J segments. The great structural diversity of TCRs is achieved by somatic recombination of these TCR gene segments during lymphocyte development in the thymus. During this process, several gene segments of each region type are randomly selected and joined to form a rearranged TCR locus. Additional junctional diversity is created by the addition or removal of nucleotides at the sites of recombination, as described in Krangel M S. Mechanics of T cell receptor gene rearrangement. Curr Opin Immunol. 2009 April; 21(2):133-9. The process of V(D)J joining plays a critical role in shaping the third hypervariable loops (also called complementary determining regions, CDR3s) of the TCR α and β chains. These regions bind antigens and are essential for providing the high specificity of antigen recognition that TCRs exhibit.

Similarly to the TCR αβ, TCR gamma (γ) and delta (δ) segments undergo V(D)J rearrangement during thymus development. Both loci are recombined in the double negative (DN) stage of T-cell development. Differentiation towards γδ or αβ lineage relies on the ability of the cell to produce functional γδ or αβ TCR. The δ locus is embedded within the α locus. Dδ, Jδ and Cδ segments are located in between the V and the J segment of the α locus. The Vδ segments are the same as the Vα segments but only a fraction of the Vα segments are used for the TCR δ chain.

Overall, V(D)J recombination is able to generate millions of different TCR sequences and plays a critical role in an organism's ability to eliminate infections or transformed cells. Not surprisingly, TCR repertoires affect a wide range of diseases, including malignancy, autoimmune disorders and infectious diseases. TCR sequencing has been instrumental for our understanding of how the TCR repertoire evolves during infection or following treatment (e.g. after hematopoietic stem cell transplantation, chronical viral infection, immunotherapy). Further, the identification of TCRs on tumor-infiltrating lymphocytes and other T-cells that target cancer-specific epitopes has not only furthered our knowledge of malignant disease, but has also led to novel therapies for cancer such as adoptive T-cell transfer or cancer vaccines.

Due to the large diversity of sequences, determining TCR repertoires has been challenging in praxis. In the last couple of years, next generation sequencing (NGS) has opened up new opportunities to comprehensively assess the extreme diversity of TCR repertoires, as described in Genolet R, Stevenson B J, Farinelli L, Oster{dot over (a)}s M, Luescher I F. Highly diverse TCRα chain repertoire of pre-immune CD8⁺ T cells reveals new insights in gene recombination. EMBO J. 2012 Apr. 4; 31(7):1666-78; Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H, Carlson C S. Comprehensive assessment of T-cell receptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107; Linnemann C, Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 November; 19(11):1534-41; Turchaninova M A, Britanova O V, Bolotin D A, Shugay M, Putintseva E V, Staroverov D B, Sharonov G, Shcherbo D, Zvyagin I V, Mamedov I Z, Linnemann C, Schumacher T N, Chudakov D M. Pairing of T-cell receptor chains via emulsion PCR. Eur J Immunol. 2013 September; 43(9):2507-15.

Since most current TCR sequencing techniques require enrichment of TCR genes for sequencing, the majority of methods include an amplification step, in which the nucleic acids encoding the individual TCRs are amplified. Therefore, one of the challenges of the TCR sequencing relates to the ability of the technology to maintain the proportion of each TCR during the amplification. Thus, the ways in which TCR libraries are prepared have a strong impact on the quality and the reliability of the obtained sequencing results and on the conclusions than can be drawn from the data. Several approaches have been used to amplify and sequence TCR repertoires in the past, each method with its own set of issues.

One frequently employed method for TCR sequencing is based on a multiplex PCR step, in which all the primers for the V and the J segments are mixed together to amplify all the possible V(D)J rearrangements/combinations, as described in Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H, Carlson C S. Comprehensive assessment of T-cell receptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107. The main drawback of this technology is that the amplification is not quantitative: Because the efficiency of each primer pair varies, some TCR sequences are preferentially represented in the library.

Another TCR sequencing method uses a process called “DNA gene capture” to isolate TCR encoding DNA fragments, as described in Linnemann C, Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 November; 19(11):1534-41. However, since this method uses DNA rather than RNA, this method will also isolate V and J segments that have not yet undergone somatic rearrangement. As a consequence, many of the obtained sequencing data are uninformative for TCR gene identification as they do not contain the V(D)J region of rearranged TCR gene locus. Furthermore, using DNA instead of RNA for the TCR gene analysis may overestimate the diversity of the TCR repertoire as only one of the two β chains is expressed by the T-cells while the other gene is silenced (allelic exclusion).

A third method of TCR amplification is based on the 5′-Race PCR technology (SMARTer® Human TCR a/b Profiling Kit, Takara-Clontech). In this method, a nucleic acid adapter is added to the 5′-end of the cDNA during the reverse transcription step. As a result, TCR products can be subsequently amplified with a single primer pair, with one primer binding to the adapter at the 5′-end of the cDNA and the second primer binding to the constant region near the 3′-end of the cDNA. One of the disadvantages of this technique is that the amplification step will generate PCR fragments ranging between 500 and 600 bp. As the length of the V segment exceeds 400 bp it is actually not possible to sequence the V(D)J junction starting from the 5′end using ILLUMINA® sequencing technology, which can generate sequencing reads of up to 300 bp only. Sequencing of the V/J junction is thus usually performed from the constant region, crossing the J segment, the CDR3 region and part of the V segment. However, sequencing errors increase with the length of the sequencing read, and are thus most frequently introduced in the V segments—the region most challenging to correctly assign due to the high homology between different V segments. Consequently, sequencing starting from the constant region may lead to a reduction in the number of V segments that can be identified unambiguously. While this caveat can be avoided by paired-end sequencing, such modification of the protocol will significantly increase the duration and cost associated with this method.

SUMMARY

With each of the current methods exhibiting significant shortcomings, there is thus a considerable need for a TCR sequencing technology that provides TCR repertoire data with high sensitivity and reliability.

Disclosed herein are methods and kits for sequencing of T-cell receptor repertoires and other immune cell repertoires, such as B-cell repertoires, with high sensitivity and reliability. In one embodiment, the methods include the steps of (1) providing RNA from T-cells, (2) transcribing RNA into complimentary RNA (cRNA), (3) reverse transcribing the cRNA into cDNA while introducing a common adapter to the 5′ end of the cDNA products, (4) amplifying the cDNA using a single primer pair, (5) further amplifying with PCR products with a single primer pair which introduces adapters for next generation sequencing, wherein the first primer binds to the common adapter region, and wherein the second primer binds to the constant region of the TCR gene, and (6) sequencing the PCR products. In one embodiment, the methods include the steps of (1) providing RNA from T-cells, (2) reverse transcribing the RNA into cDNA, (3) generating second strand cDNA while introducing a common adapter to the 5′ end of the cDNA products, (4) amplifying the cDNA using a single primer pair, (5) further amplifying with PCR products with a single primer pair which introduces adapters for next generation sequencing, wherein the first primer binds to the common adapter region, and wherein the second primer binds to the constant region of the TCR gene, and (6) sequencing the PCR products. These embodiments are also called SEQTR method (Sequencing T-cell Receptors). Also provided are kits containing primer mixtures for the sequencing of T-cell receptor repertoires. Similar methods and kits for sequencing of B-cell receptor repertoires are provided.

According to one aspect, methods for sequencing immune cell receptor genes are provided. The methods include (1) providing RNA from immune cells; 2)(a) optionally transcribing the RNA into complementary RNA (cRNA), followed by reverse transcribing the cRNA into complementary DNA (cDNA) using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by reverse transcription contains the first adapter sequence; (2)(b) if step (2)(a) is not performed, reverse transcribing the RNA into complementary DNA (cDNA), followed by transcribing the cDNA into second strand cDNA using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by transcribing the cDNA into second strand cDNA contains the first adapter sequence; (3) amplifying the cDNA to produce a first amplification product using a first primer pair comprising a first primer that hybridizes to the first adapter sequence and a second primer that hybridizes to a constant region of immune cell receptor gene; (4) amplifying the first amplification product to produce a second amplification product using a second primer pair, in which (i) a first primer of the second primer pair binds to the adapter sequence at the 5′ end of the second amplification product, (ii) the second primer of the second primer pair binds to the constant region of immune cell receptor gene in the second amplification product, and (iii) the first and second primers comprise adapter sequences for sequencing; and (5) sequencing the second amplification product.

In some embodiments, the reverse transcription step results in PCR products ranging from 150-600 bp. In some embodiments, the immune cell receptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR) genes.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR α chain V segments. In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR β chain V segments. In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR γ chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR δ chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR heavy chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR light chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) contain a nucleotide barcode sequence. In some embodiments, the nucleotide barcode comprises 6 to 20 nucleotides. In some embodiments, the nucleotide barcode consists of 9 nucleotides. In some embodiments, the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN or HHHHHNNNN.

In some embodiments, the first adapter sequence of the one or more primers used for the reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprises a T7 adapter or an ILLUMINA® adapter.

In some embodiments, the immune cells are T-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a TCR gene.

In some embodiments, the immune cells are B-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a BCR gene.

In some embodiments, the sequencing is next generation sequencing.

In some embodiments, the RNA from the immune cells is obtained by mixing immune cells with carrier cells before RNA extraction.

In some embodiments, the immune cells are tumor-infiltrating lymphocytes.

In some embodiments, the immune cells are CD4 or CD8 positive T-cells.

In some embodiments, the immune cells are purified from peripheral blood mononuclear cells (PBMC) before RNA extraction.

In some embodiments, the immune cells are part of a mixture of PBMC.

In some embodiments, the immune cells are derived from a mammal. In some embodiments, the mammal is a human or a mouse.

According to another aspect, kits for sequencing of T-cell receptors are provided. The kits include at least one primer which comprises a TCR α chain V segment portion of any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310 and a barcode sequence. In some embodiments, the kits include at least one primer including any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.

According to another aspect, kits for sequencing of T-cell receptors are provided. The kits include at least one primer which comprises a TCR β chain V segment portion of any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360 and a barcode sequence. In some embodiments, the kits include at least one primer comprising any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the arrangement of (variable) V, diversity (D), joining (J) and constant (C) regions in the α and β chains of T-cell receptors. Figure taken from Murphy, K., Travers, P., Walport, M., & Janeway, C. (2012). Janeway's immunobiology. New York: Garland Science.

FIG. 2 is an illustration of three different TCR sequencing techniques that have been employed in the past.

FIG. 3 provides an overview of the SEQTR method, using TCR α chains as an example. Each bar represents a TCR α chain gene. In RNA and cRNA molecules, the order of the segments is, left to right: V segments, J segments, and the constant region. Barcode regions are added in cDNA molecule to the left of V segments; and T7 adapter regions are added to the left of the barcodes (also indicated by T7 primer amplification in PCR1 and PCR2 steps). ILLUMINA® sequencing adapters are added in the PCR2 step to the 5′ and 3′ ends of the molecules, as shown in the last set of molecules.

FIG. 4 illustrates the sensitivity of the SEQTR method. 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, were mixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extracted and subjected to transcription, reverse transcription and one round of amplification (steps 2-4, see Detailed Description). The resulting PCR products were separated on an agarose gels and visualized with ethidium bromide.

FIG. 5 illustrates the specificity of the SEQTR method. 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, were mixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extracted and subjected to the SEQTR method. The percentages of sequencing reads that were or were not, respectively, associated with actual TCR genes are indicated.

FIG. 6 illustrates the unambiguous identification of TCR genes as a feature of the SEQTR method. 5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to the SEQTR method. Reads that were not associated with TCR genes were removed from the data set. For the remaining reads, the percentages of reads that could or could not, respectively, be unambiguously assigned to specific V or J segments are indicated.

FIG. 7 illustrates the linearity of the SEQTR method. A fixed amount of DNA encoding a known TCR sequence was diluted at different concentrations into a DNA mixture representing a naïve CD8 T-cell repertoire. TCR repertoires of the individual mixtures were sequenced using the SEQTR method. The observed frequency of the known TCR sequence in the entire repertoire was plotted against the respective TCR gene dilution.

FIG. 8 illustrates the reproducibility of the SEQTR method. TCR repertoires were sequenced using the SEQTR method from one biological sample in two independent technical replicates. The frequencies for each V-J rearrangement/combination in TCR β chains were determined and compared between the two replicates. Each sphere represents a single V-J rearrangement with the size of a sphere indicating the relative frequency of the specific V-J recombination. Grey spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by less than two-fold. Black spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by more than two-fold.

FIGS. 9A-9C illustrates the diversity of three different TCR repertoire sequencing data set using the SEQTR method. FIG. 9A CD8 positive T-cells were isolated from peripheral blood mononuclear cells (PBMC), FIG. 9B additionally purified using tetramers conjugated with neo-epitope TEDYMIHII (SEQ ID NO:236) or FIG. 9C additionally purified using tetramer conjugated with neo-epitope (same as in FIG. 9B) and subsequently expanded in vitro. The TCR repertoires of the respective samples were sequenced using the SEQTR method and the relative frequencies of all observed V/J rearrangements/combinations plotted.

FIGS. 10A-10C illustrate the overlap of TCRs identified using a single cell cloning method and TCRs identified using the SEQTR method. FIG. 10A: T-cells were isolated from PBMC and subjected to an additional round of purification using tetramers conjugated with neo-epitope TEDYMIHII (SEQ ID NO: 236). The resulting cell population was then sorted by fluorescence-activated cell sorting (FACS). Half of the sorted cells were subjected to the SEQTR method to sequence the TCR repertoire. For the other half of cells, individual T-cell clones were isolated and expanded in vitro (single cell cloning). Once the clones were established, the TCR genes of each T-cell clones were amplified and sequenced using classical Sanger sequencing. FIG. 10B: The table shows all six TCRs identified using the single cell cloning method. The sequences correspond to SEQ ID NOs: 237 through 242 from top to bottom, respectively. FIG. 10C: The table shows the eight most frequent TCRs identified using the SEQTR method. The sequences correspond to SEQ ID NOs: 243 through 250 from top to bottom, respectively.

FIG. 11 illustrates the number of reads for different samples obtained using the TCR sequencing service “immunoSEQ®” offered by Adaptive Biotechnology. The requested number of reads per sample was 200,000 reads. The number on the x axis represent analysis of samples from 16 different patients. Columns, left to right, for each sample represent number of reads from: the tumor; the stroma (tissue surrounding the tumor); epitope specific TIL (Tumor Infiltrating Lymphocyte) stained with tetramer and sorted by FACS from the tumor sample (TET); and tetramer sorted TIL from a piece of the tumor that has been engrafted in a mice (mTET).

FIG. 12 illustrates the amplification of TCR genes from T-cells that are part of a PBMC mixture (upper panel) or from isolated, CD4 positive T-cells (lower panel), using steps 2 to 4 of the SEQTR method.

DETAILED DESCRIPTION

In light of the shortcomings of existing techniques to sequence TCRs, it was determined that a TCR sequencing technology providing the most reliable TCR repertoire data includes the following features:

• • 1) The amplification of TCR genes is linear and does not employ multiplex PCR, therefore avoiding artificial overrepresentation of certain TCR sequences.
  • 2) The method is based on RNA and not DNA, thus only providing data for TCR sequences that have undergone rearrangement and that are actually expressed in T-cells.
  • 3) TCR genes are sequenced from the 5′ end, providing high quality sequencing data and therefore maximizing reliable and unambiguous identification of the highly homologous V segments.
  • 4) Sequencing data include the highly variable CDR3 region, therefore facilitating unambiguous identification of TCR sequences.

The disclosed methods, systems and kits fulfill all these criteria. These same features are of use in sequencing receptors from other immune cells, such as B-cells.

In some embodiments, the immune cell receptor sequencing methods comprise the following steps:

• • (1) Providing total RNA (RNA) as the starting material;
  • (2)(a) Transcribing the RNA into complimentary RNA (cRNA) followed by reverse transcribing the cRNA into cDNA, using primers that introduce a common adapter to the 5′ end of the cDNA products;
  • (2)(b) If step (2)(a) is not performed, reverse transcribing the RNA into complementary DNA (cDNA), followed by transcribing the cDNA into second strand cDNA using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by transcribing the cDNA into second strand cDNA contains the first adapter sequence;
  • (3) Amplifying the cDNA products using a single primer pair;
  • (4) Amplifying the PCR products of step 4 using a single primer pair, in which:

i. the primers introduce adapters for next generation sequencing, and ii. the first primer binds to the common adapter region at the 5′ end of the PCR products, and iii. the second primer binds to a region of the PCR products that constitutes the constant region of the TCR to be sequenced; and

• • (5) Sequencing the PCR products generated in step 4.

Genetic Information to be Sequenced

The genetic information to be sequenced is immune cell receptor genes. In the some embodiments of the invention, the genetic information to be sequenced comprises T-cell receptors genes. In some embodiments, the TCR genes that are sequenced encode TCR α chains or TCR β chains. In other embodiments, TCR genes that are sequenced encode TCR δ chains or TCR γ chains.

In other embodiments of the invention, the genetic information to be sequenced comprises B-cell receptor (BCR) genes.

Starting Material (Step 1)

RNA is isolated from immune cells and used to generate complimentary RNA (cRNA) by in vitro transcription. This is in contrast to existing TCR sequencing techniques that use DNA or complementary DNA (cDNA) as their genetic starting material.

In some embodiments, the immune cells from which RNA is obtained are isolated from peripheral blood mononuclear cells before RNA extraction. The immune cells are, in some embodiments, T-cells or B-cells.

In some embodiments, T-cells from which RNA is obtained express CD4 or CD8.

Generation of cRNA Through Transcription (Step (2)(a))

Complementary RNA (cRNA) is generated by in vitro transcription. Any method for performing in vitro transcription known to those skilled in molecular biology can be used. In some embodiments, the in vitro transcription in step 2 is performed using commercially available kits, such as the AMBION™ kits available from Thermo Fisher Scientific.

Reverse Transcription (Step (2)(a))

Reverse transcription of the cRNA is performed to generate complementary DNA (cDNA). Methods known to persons skilled in molecular biology are used to reverse transcribe cRNA to cDNA. Typically, such methods include hybridization of a primer to the 3′ end of the cRNA molecule and production of DNA starting at the hybridized primer using a reverse transcriptase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the reverse transcription reaction is important for the ability to differentiate between homologous, yet distinct, immune cell receptor sequences with high degrees of certainty and allows shortening of the V segments from the 5′ end, generating PCR products with a size of 250-300 bp. Such a size range of PCR products is optimal for next generation sequencing.

In some embodiments, the primers used for the reverse transcription are designed to bind within the V segments of the TCR genes (see FIG. 3). For example, the reverse transcription primers are designed to bind close enough to the V(D)J junction so that the resulting sequencing data cover the CDR3 of the V segment and the J segment, but far enough from the V(D)J junction to still allow differentiation between different V regions.

In some embodiments of the invention, a set of preferred primers is used (see, e.g., the sequences in Table 2 and Table 4, and Table 8 and Table 9). Due to the high degree of homology between different V segments, some of the primers described in Table 2 and Table 4 (and Table 8 and Table 9) bind to more than one V segment (see Table 3 and Table 5; the binding sites in their respective V segments for primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 are indicated in Table 15 and Table 16). However, the design of the primers presented in Table 2 and Table 4 (likewise Table 8 and Table 9) still allows the unambiguous assignment/identification of the respective V segments based on differences between the V segments downstream of the primer-binding site. In an alternative embodiment of the invention, only a subset of the preferred primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 may be used for the reverse transcription.

In yet another embodiment of the invention, primer sets may be used that bind to different regions in the V segments when compared to the primers having SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. For instance, the binding site of one or more primers may be moved towards the CDR3 region of the TCR gene. Due to the high degree of homology between V segments, the further the primer binding site is moved in the direction of the CDR3 region of the TCR gene, the larger the likelihood that the resulting sequencing data are consistent with the presence of more than one V segment. While, in these cases, the respective V segments cannot be assigned or identified unambiguously, the number of V/J segments possibly present in the sample can often be narrowed down to a small subset. Depending on the application, such limited information can already be of value to the experimenter.

In another embodiment of the invention, the binding site of one or more primers may be moved towards the 5′ end of the V segment as compared to the binding sites of primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. Many next generation sequencing technologies generate sequencing reads that are 150 bp long. Therefore, the further the primer binding site is moved towards the 5′ end of the V segment, the larger is the probability that the respective J segment (which can be found at the 3′ end of the resulting sequencing read) cannot be identified unambiguously. However, this problem can be circumvented by using alternative sequencing technologies that generate reads >150 bp.

In some embodiments, the primers used in step (2)(a) additionally contain a unique bar code. Such barcoding of each RNA molecule before the amplification can be used to correct the obtained sequencing results for PCR and sequencing errors.

In some embodiments, the primers for this reverse transcription step introduce a common T7 adapter at the 5′ end of the resulting PCR products. However, alternative adapter sequences are possible, including, but not limited to ILLUMINA® adapters and sequences presented in Table 1.

TABLE 1
Examples for alternative nucleotide adapters that can
be used instead of a T7 adapter sequence

SEQ ID NO	Primer name	Primer sequence (5′ to 3′)
251	Original Eberwine T7	AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCT
252	Affymetrix T7	GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGT
253	Invitrogen T7	TAATACGACTCACTATAGGGAGGCGGT
254	Ambion T7	GGTAATACGACTCACTATAGGGAGAAGAGT
255	Agilent T7	AATTAATACGACTCACTATAGGGAGAT

Reverse Transcription (Step (2)(b))

Reverse transcription of the RNA is performed to generate complementary DNA (cDNA). Methods known to persons skilled in molecular biology are used to reverse transcribe RNA to cDNA. Typically, such methods include hybridization of a primer to the 3′ end of the RNA molecule and production of DNA starting at the hybridized primer using a reverse transcriptase enzyme and appropriate nucleotides, salts and buffers.

Transcribing the cDNA into Second Strand cDNA (Step (2)(b))

Following generation of cDNA, second strand cDNA is synthesized using methods known to persons skilled in molecular biology. Typically, such methods include hybridization of a primer to the 3′ end of the cDNA molecule and production of second strand cDNA starting at the hybridized primer using a polymerase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the second strand synthesis reaction is step (2)(b) is as described above for reverse transcription in step (2)(a). The choice of primers is important for the ability to differentiate between homologous, yet distinct, immune cell receptor sequences with high degrees of certainty and allows shortening of the V segments from the 5′ end, generating PCR products with a size of 250-300 bp. Such a size range of PCR products is optimal for next generation sequencing.

Amplification (Step 3)

Amplification of the cDNA is performed by any of the well-known amplification reactions, such as polymerase chain reaction (PCR). Methods known to persons skilled in the molecular biology art are used to amplify the cDNA or a portion thereof (e.g., as depicted in FIG. 3). Typically, such methods include hybridization of a pair of primers to the cDNA molecule and amplification of the DNA sequence between the hybridized primers using a polymerase enzyme and appropriate nucleotides, salts and buffers.

In some embodiments, the first primer of a primer pair used in an amplification step binds to the common adapter region of the cDNA products produced in step 3 and the second primer of the primer pair binds to a region of the cDNA products that constitutes the constant region of the TCR to be sequenced (see FIG. 3).

Of note, not all reverse primers designed to target the constant region of the TCR gene perform equally well in this reaction. For example, the primers listed in Table 7 all failed to provide good amplification with the selected T7 5′ adapter. Therefore, in certain embodiments, the primers listed in are Table 6 used in this amplification step.

Amplification (Step 4)

A second amplification step is performed to add additional sequences to the amplified molecules, such as sequences that are useful in downstream DNA sequencing reactions. In some embodiments of the present invention, the primers used in this step add appropriate adapters for ILLUMINA® sequencing.

Sequencing (Step 5)

Various suitable sequencing methods described herein or known in the art are used to obtain sequence information from the amplified sequences from the nucleic acid molecules within a sample. For example, sequencing methodologies that can be used in the methods disclosed herein include: classic Sanger sequencing, massively parallel sequencing, next generation sequencing, polony sequencing, 454 pyrosequencing, ILLUMINA® sequencing, SOLEXA® sequencing, SOLID™ sequencing (sequencing by oligonucleotide ligation and detection), ion semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time sequencing, nanopore DNA sequencing, tunneling currents DNA sequencing, sequencing by hybridization, sequencing with mass spectrometry, microfluidic Sanger sequencing, microscopy-based sequencing, RNA polymerase sequencing, in vitro virus high-throughput sequencing, Maxam-Gilbert sequencing, single-end sequencing, paired-end sequencing, deep sequencing, and/or ultra-deep sequencing.

Definitions

As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

As used herein, a “primer” is a nucleic acid molecule that hybridizes with a complementary (including partially complementary) polynucleotide strand. Primers can be DNA molecules, RNA molecules, or DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs.

EXAMPLES

Example 1: Exemplary Protocol for the SEQTR Method Using T7 and TrueSeq Adapters

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting material and RNA extraction

• • To obtain sufficient amounts of RNA in the extraction, a minimum of 500,000 T-cells were used as starting material. Alternatively, and especially in instances where fewer T-cells were available, T-cells were mixed with 50,000 mouse 3T3 cells that served as carrier. T-cell RNA was extracted using the RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's instruction with the following modification: Elution was performed with 20 μl of water preheated to 50° C. RNA quality and quantity was verified using a fragment analyzer.

2) cRNA synthesis by in vitro transcription (IVT):

• • In vitro transcription of isolated RNA was performed using the MessageAmp™ II aRNA Amplification Kit from Ambion® (Thermo Fisher Scientific), which contains enzymes, buffers and nucleotides required to perform the first and second strand cDNA and the in vitro transcription. The kit also provides all columns and reagents needed for the cDNA and cRNA purifications. RNA amplification was performed according to the manufacturer's instructions with the following modifications: 1) Between 0.5 and 1 μg of total RNA as was used as starting material. 2) The IVT was performed in a final volume of 40 μl, and incubated at 37° C. for 16 h. Purified cRNA was quantified by absorbance using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific).

3) cDNA synthesis by reverse transcription:

• • The reverse transcription of the cRNA was performed with the SuperScript® III from Invitrogen (Thermo Fisher Scientific). The kit provides the enzyme, the buffer and the dithiothreitol (DTT) needed for the reaction. Deoxynucleotides (dNTPs) and RNAsin® Ribonuclease inhibitor were purchased from Promega. The sequences for the primers used for the reverse transcription can be found in Table 2 (primers for sequencing TCR α chain genes) and Table 4 (primers for sequencing TCR β chain genes).
  • 500 ng of cRNA were used as starting material for the reverse transcription. cRNA was mixed with 1 μl hTRAV or hTRBV primers mix (2 μM each) and 1 μl dNTP (25 mM) in a final volume of 13 μl. The mix was first incubated at 70° C. for 10 min, then at 50° C. for 30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μg SuperScript III and 1 μl RNAsin® were added to the mix. The samples were subsequently incubated for at 55° C. 1 h and then at 85° C. for 5 min. After the cDNA synthesis, 1 μg DNase-free RNase (Roche) was added to the cDNA and incubated at 37° C. for 30 min to remove the cRNA.

4) TCR gene amplification:

• • TCR gene amplification was performed using a Phusion® High-Fidelity DNA polymerase (New England Biolabs) under the following conditions: PCR mix: 1 μl cDNA from step 3, 1 μl dNTPs (25 mM), 1 μl primer mix (10 μM each, see Table 5), 5 μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume of 25 μl.
  • PCR conditions:
    • 94° C. for 5 min
    • 20 to 30 cycles of
      • 98° C. for 10 s
      • 55° C. for 30 s
      • 72° C. for 30 s
    • 72° C. for 2 min
  • PCR products were purified either from agarose gels (using a Qiaquick Gel Extraction Kit from Qiagen) or using an ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) according to the manufacturer's instructions.

5) Addition of Next Generation Sequencing adapters:

• • ILLUMINA® sequencing adapters were added by PCR using a Phusion® High-Fidelity DNA polymerase (New England Biolabs). One third of the purified PCR product obtained in step 4 was mixed with 0.5 μl dNTPs (25 mM), 1 μl primer mix (10 μM each, see Table 8), 5 μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume of 25 μl.
  • PCR conditions:
    • 94° C. for 5 min
    • perform 12 cycles of:
      • 98° C. for 10 s
      • 55° C. for 30 s
      • 72° C. for 30 s
    • 72° C. for 2 min

6) TCR library purification:

• • 10 μl of the PCR product from step 5 were purified using an ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) or Ampure XP beads (Beckman Coulter) according to the manufacturer's instruction. Samples could then directly be used for ILLUMINA® sequencing.

TABLE 2
Preferred primer sequences for amplification of TCR a chain V segments. N can be any
nucleotide. The sequences for primers presented in this table consist of three parts
(listed from 5′ to 3′): T7 adapter, barcode and TCR a chain V segment.

			Sequence
SEQ		Sequence T7 adapter	barcode portion	Sequence TCR a chain V
ID NO	Primer name	portion of the primer	of the primer	segment portion of the primer

1	hTRAV1-1	TGTAATACGACTCACTATAG	NNNNTNNNN	CTTCTACAGGAGCTCCAGATGAAAG
2	hTRAV1-2	TGTAATACGACTCACTATAG	NNNNTNNNN	CTTTTGAAGGAGCTCCAGATGAAAG
3	hTRAV2	TGTAATACGACTCACTATAG	NNNNTNNNN	TGCTCATCCTCCAGGTGCGGGA
4	hTRAV3	TGTAATACGACTCACTATAG	NNNNTNNNN	GAAGAAACCATCTGCCCTTGTGA
5	hTRAV4	TGTAATACGACTCACTATAG	NNNNTNNNN	CCTGCCCCGGGTTTCCCTGAGCGAC
6	hTRAV5	TGTAATACGACTCACTATAG	NNNNTNNNN	TCTCTGCGCATTGCAGACACCCA
7	hTRAV6	TGTAATACGACTCACTATAG	NNNNTNNNN	TTGTTTCATATCACAGCCTCCCA
8	hTRAV7	TGTAATACGACTCACTATAG	NNNNTNNNN	GCTTGTACATTACAGCCGTGCA
9	hTRAV8-1/8-3	TGTAATACGACTCACTATAG	NNNNTNNNN	ATCTGAGGAAACCCTCTGTGCA
10	hTRAV8-2/8-4	TGTAATACGACTCACTATAG	NNNNTNNNN	ACCTGACGAAACCCTCAGCCCAT
11	hTRAV8-5	TGTAATACGACTCACTATAG	NNNNTNNNN	CCTATGCCTGTCTTTACTTTAATC
12	hTRAV8-6	TGTAATACGACTCACTATAG	NNNNTNNNN	CTTGAGGAAACCCTCAGTCCATAT
13	hTRAV8-7	TGTAATACGACTCACTATAG	NNNNTNNNN	GAAACCATCAACCCATGTGAGTGA
14	hTRAV9-1	TGTAATACGACTCACTATAG	NNNNTNNNN	ACTTGGAGAAAGACTCAGTTCAA
15	hTRAV9-2	TGTAATACGACTCACTATAG	NNNNTNNNN	ACTTGGAGAAAGGCTCAGTTCAA
16	hTRAV10	TGTAATACGACTCACTATAG	NNNNTNNNN	CTGCACATCACAGCCTCCCA
17	hTRAV11	TGTAATACGACTCACTATAG	NNNNTNNNN	GTTTGGAATATCGCAGCCTCTCAT
18	hTRAV12-1	TGTAATACGACTCACTATAG	NNNNTNNNN	CCCTGCTCATCAGAGACTCCAAG
19	hTRAV12-2	TGTAATACGACTCACTATAG	NNNNTNNNN	CTCTGCTCATCAGAGACTCCCAG
20	hTRAV12-3	TGTAATACGACTCACTATAG	NNNNTNNNN	CCTTGTTCATCAGAGACTCACAG
21	hTRAV13-1	TGTAATACGACTCACTATAG	NNNNTNNNN	TCCCTGCACATCACAGAGACCCAA
22	hTRAV13-2	TGTAATACGACTCACTATAG	NNNNTNNNN	TCTCTGCAAATTGCAGCTACTCAA
23	hTRAV14	TGTAATACGACTCACTATAG	NNNNTNNNN	TTGTCATCTCCGCTTCACAACTGG
24	hTRAV15	TGTAATACGACTCACTATAG	NNNNTNNNN	GTTTTGAATATGCTGGTCTCTCAT
25	hTRAV16	TGTAATACGACTCACTATAG	NNNNTNNNN	CCTGAAGAAACCATTTGCTCAAGA
26	hTRAV17	TGTAATACGACTCACTATAG	NNNNTNNNN	TCCTTGTTGATCACGGCTTCCCGG
27	hTRAV18	TGTAATACGACTCACTATAG	NNNNTNNNN	ACCTGGAGAAGCCCTCGGTGCA
28	hTRAV19	TGTAATACGACTCACTATAG	NNNNTNNNN	CACCATCACAGCCTCACAAGTCGT
29	hTRAV20	TGTAATACGACTCACTATAG	NNNNTNNNN	TTTCTGCACATCACAGCCCCTA
30	hTRAV21	TGTAATACGACTCACTATAG	NNNNTNNNN	CTTTATACATTGCAGCTTCTCAGCC
31	hTRAV22	TGTAATACGACTCACTATAG	NNNNTNNNN	GTACATTTCCTCTTCCCAGACCAC
32	hTRAV23	TGTAATACGACTCACTATAG	NNNNTNNNN	CATTGCATATCATGGATTCCCAGC
33	hTRAV24	TGTAATACGACTCACTATAG	NNNNTNNNN	GCTATTTGTACATCAAAGGATCCC
34	hTRAV25	TGTAATACGACTCACTATAG	NNNNTNNNN	CAGCTCCCTGCACATCACAGCCA
35	hTRAV26-1	TGTAATACGACTCACTATAG	NNNNTNNNN	TTGATCCTGCCCCACGCTACGCTGA
36	hTRAV26-2	TGTAATACGACTCACTATAG	NNNNTNNNN	TTGATCCTGCACCGTGCTACCTTGA
37	hTRAV27	TGTAATACGACTCACTATAG	NNNNTNNNN	GTTCTCTCCACATCACTGCAGCC
38	hTRAV28	TGTAATACGACTCACTATAG	NNNNTNNNN	GCCACCTATACATCAGATTCCCA
39	hTRAV29	TGTAATACGACTCACTATAG	NNNNTNNNN	TCTCTGCACATTGTGCCCTCCCA
40	hTRAV30	TGTAATACGACTCACTATAG	NNNNTNNNN	CCCTGTACCTTACGGCCTCCCAGCT
41	hTRAV31	TGTAATACGACTCACTATAG	NNNNTNNNN	CTTATCATATCATCATCACAGCCA
42	hTRAV32	TGTAATACGACTCACTATAG	NNNNTNNNN	TCCCTGCATATTACAGCCACCCAA
43	hTRAV33	TGTAATACGACTCACTATAG	NNNNTNNNN	ACCTCACCATCAATTCCTTAAAAC
44	hTRAV34	TGTAATACGACTCACTATAG	NNNNTNNNN	TCCCTGCATATCACAGCCTCCCAG
45	hTRAV35	TGTAATACGACTCACTATAG	NNNNTNNNN	CTTCCTGAATATCTCAGCATCCAT
46	hTRAV36	TGTAATACGACTCACTATAG	NNNNTNNNN	TCCTGAACATCACAGCCACCCAG
47	hTRAV37	TGTAATACGACTCACTATAG	NNNNTNNNN	TCCCTGCACATACAGGATTCCCAG
48	hTRAV38	TGTAATACGACTCACTATAG	NNNNTNNNN	CAAGATCTCAGACTCACAGCTGG
49	hTRAV39	TGTAATACGACTCACTATAG	NNNNTNNNN	CCGTCTCAGCACCCTCCACATCA
50	hTRAV40	TGTAATACGACTCACTATAG	NNNNTNNNN	CCATTGTGAAATATTCAGTCCAGG

TABLE 3
V segments targeted by each primer used for
the amplification of TCR α chain V segments.

SEQ
ID NO	Primer	Targeted V segment(s)

1	hTRAV1-1	hTRAV01-1
2	hTRAV1-2	hTRAV01-2
3	hTRAV2	hTRAV02
4	hTRAV3	hTRAV03
5	hTRAV4	hTRAV04
6	hTRAV5	hTRAV05
7	hTRAV6	hTRAV06
8	hTRAV7	hTRAV07
9	hTRAV8-1/8-3	hTRAV08-1, hTRAV08-3
10	hTRAV8-2/8-4	hTRAV08-2, hTRAV08-4
11	hTRAV8-5	hTRAV08-5
12	hTRAV8-6	hTRAV08-6
13	hTRAV8-7	hTRAV08-7
14	hTRAV9-1	hTRAV09-1
15	hTRAV9-2	hTRAV09-2
16	hTRAV10	hTRAV10, hTRAV41
17	hTRAV11	hTRAV11
18	hTRAV12-1	hTRAV12-1
19	hTRAV12-2	hTRAV12-2
20	hTRAV12-3	hTRAV12-3
21	hTRAV13-1	hTRAV13-1
22	hTRAV13-2	hTRAV13-2
23	hTRAV14	hTRAV14
24	hTRAV15	hTRAV15
25	hTRAV16	hTRAV16
26	hTRAV17	hTRAV17
27	hTRAV18	hTRAV18
28	hTRAV19	hTRAV19
29	hTRAV20	hTRAV20
30	hTRAV21	hTRAV21
31	hTRAV22	hTRAV22
32	hTRAV23	hTRAV23
33	hTRAV24	hTRAV24
34	hTRAV25	hTRAV25
35	hTRAV26-1	hTRAV26-1
36	hTRAV26-2	hTRAV26-2
37	hTRAV27	hTRAV27
38	hTRAV28	hTRAV28
39	hTRAV29	hTRAV29
40	hTRAV30	hTRAV30
41	hTRAV31	hTRAV31
42	hTRAV32	hTRAV32
43	hTRAV33	hTRAV33
44	hTRAV34	hTRAV34
45	hTRAV35	hTRAV35
46	hTRAV36	hTRAV36
47	hTRAV37	hTRAV37
48	hTRAV38	hTRAV38-1, hTRAV38-2
49	hTRAV39	hTRAV39
50	hTRAV40	hTRAV40

TABLE 4
Preferred primer sequences for amplification of TCR 3 chain V segments. N can be any
nucleotide. The sequences for primers presented in this table consist of three parts
(listed from 5′ to 3′): T7 adapter, barcode and TCR 3 chain V segment.

			Sequence
SEQ		Sequence T7 adapter	barcode portion	Sequence TCR 3 chain V segment
ID NO	Primer name	portion of the primer	of the primer	portion of the primer

51	hTRBV1	TGTAATACGACTCACTATAG	NNNNANNNN	GTGGTCGCACTGCAGCAAGAAGA
52	hTRBV2	TGTAATACGACTCACTATAG	NNNNANNNN	GATCCGGTCCACAAAGCTGGAGGA
53	hTRBV3-1	TGTAATACGACTCACTATAG	NNNNANNNN	CATCAATTCCCTGGAGCTTGGTGA
54	hTRBV4-1	TGTAATACGACTCACTATAG	NNNNANNNN	TTCACCTACACGCCCTGCAGCCAG
55	hTRBV4-2	TGTAATACGACTCACTATAG	NNNNANNNN	TTCACCTACACACCCTGCAGCCAG
56	hTRBV5-1	TGTAATACGACTCACTATAG	NNNNANNNN	GAATGTGAGCACCTTGGAGCTGG
57	hTRBV5-2	TGTAATACGACTCACTATAG	NNNNANNNN	TACTGAGTCAAACACGGAGCTAGG
58	hTRBV5-3	TGTAATACGACTCACTATAG	NNNNANNNN	GCTCTGAGATGAATGTGAGTGCCT
59	hTRBV5-4	TGTAATACGACTCACTATAG	NNNNANNNN	CTGAGCTGAATGTGAACGCCTT
60	hTRBV6-1	TGTAATACGACTCACTATAG	NNNNANNNN	GAGTTCTCGCTCAGGCTGGAGT
61	hTRBV6-2	TGTAATACGACTCACTATAG	NNNNANNNN	CTGGGGTTGGAGTCGGCTGCTC
62	hTRBV6-4	TGTAATACGACTCACTATAG	NNNNANNNN	CCCCTCACGTTGGCGTCTGCTG
63	hTRBV6-5	TGTAATACGACTCACTATAG	NNNNANNNN	TCCCGCTCAGGCTGCTGTCGGC
64	hTRBV6-6	TGTAATACGACTCACTATAG	NNNNANNNN	GATTTCCCGCTCAGGCTGGAGT
65	hTRBV6-7	TGTAATACGACTCACTATAG	NNNNANNNN	TCCCCCTCAAGCTGGAGTCAGCT
66	hTRBV6-8	TGTAATACGACTCACTATAG	NNNNANNNN	TCCCACTCAGGCTGGTGTCGGC
67	hTRBV7-1	TGTAATACGACTCACTATAG	NNNNANNNN	CTCTGAAGTTCCAGCGCACACA
68	hTRBV7-2	TGTAATACGACTCACTATAG	NNNNANNNN	GATCCAGCGCACACAGCAGGAG
69	hTRBV7-3	TGTAATACGACTCACTATAG	NNNNANNNN	ACTCTGAAGATCCAGCGCACAGA
70	hTRBV7-5	TGTAATACGACTCACTATAG	NNNNANNNN	AGATCCAGCGCACAGAGCAAGG
71	hTRBV7-6	TGTAATACGACTCACTATAG	NNNNANNNN	CAGCGCACAGAGCAGCGGGACT
72	hTRBV7-9	TGTAATACGACTCACTATAG	NNNNANNNN	GAGATCCAGCGCACAGAGCAGG
73	hTRBV8-1	TGTAATACGACTCACTATAG	NNNNANNNN	CCCTCAACCCTGGAGTCTACTA
74	hTRBV8-2	TGTAATACGACTCACTATAG	NNNNANNNN	TCCCCAATCCTGGCATCCACCA
75	hTRBV9	TGTAATACGACTCACTATAG	NNNNANNNN	CTAAACCTGAGCTCTCTGGAGCT
76	hTRBV10-1	TGTAATACGACTCACTATAG	NNNNANNNN	CCCTCACTCTGGAGTCTGCTGC
77	hTRBV10-2	TGTAATACGACTCACTATAG	NNNNANNNN	CCCTCACTCTGGAGTCAGCTAC
78	hTRBV10-3	TGTAATACGACTCACTATAG	NNNNANNNN	TCCTCACTCTGGAGTCCGCTAC
79	hTRBV11-1	TGTAATACGACTCACTATAG	NNNNANNNN	CCACTCTCAAGATCCAGCCTGCA
80	hTRBV12-1	TGTAATACGACTCACTATAG	NNNNANNNN	GAGGATCCAGCCCATGGAACCCA
81	hTRBV12-2	TGTAATACGACTCACTATAG	NNNNANNNN	CTGAAGATCCAGCCTGCAGAGC
82	hTRBV12-3	TGTAATACGACTCACTATAG	NNNNANNNN	CAGCCCTCAGAACCCAGGGACT
83	hTRBV13	TGTAATACGACTCACTATAG	NNNNANNNN	GAGCTCCTTGGAGCTGGGGGACT
84	hTRBV14	TGTAATACGACTCACTATAG	NNNNANNNN	GGTGCAGCCTGCAGAACTGGAG
85	hTRBV15	TGTAATACGACTCACTATAG	NNNNANNNN	GACATCCGCTCACCAGGCCTGG
86	hTRBV16	TGTAATACGACTCACTATAG	NNNNANNNN	TGAGATCCAGGCTACGAAGCTT
87	hTRBV17	TGTAATACGACTCACTATAG	NNNNANNNN	GAAGATCCATCCCGCAGAGCCG
88	hTRBV18	TGTAATACGACTCACTATAG	NNNNANNNN	GGATCCAGCAGGTAGTGCGAGG
89	hTRBV19	TGTAATACGACTCACTATAG	NNNNANNNN	CACTGTGACATCGGCCCAAAAG
90	hTRBV20	TGTAATACGACTCACTATAG	NNNNANNNN	CTGACAGTGACCAGTGCCCATC
91	hTRBV21	TGTAATACGACTCACTATAG	NNNNANNNN	GAGATCCAGTCCACGGAGTCAG
92	hTRBV22	TGTAATACGACTCACTATAG	NNNNANNNN	GTGAAGTTGGCCCACACCAGCCA
93	hTRBV23	TGTAATACGACTCACTATAG	NNNNANNNN	CCTGGCAATCCTGTCCTCAGAA
94	hTRBV24	TGTAATACGACTCACTATAG	NNNNANNNN	GAGTCTGCCATCCCCAACCAGA
95	hTRBV25	TGTAATACGACTCACTATAG	NNNNANNNN	GGAGTCTGCCAGGCCCTCACA
96	hTRBV26	TGTAATACGACTCACTATAG	NNNNANNNN	GAAGTCTGCCAGCACCAACCAG
97	hTRBV27	TGTAATACGACTCACTATAG	NNNNANNNN	GGAGTCGCCCAGCCCCAACCAG
98	hTRBV28	TGTAATACGACTCACTATAG	NNNNANNNN	GGAGTCCGCCAGCACCAACCAG
99	hTRBV29	TGTAATACGACTCACTATAG	NNNNANNNN	GTGAGCAACATGAGCCCTGAAGA
100	hTRBV30	TGTAATACGACTCACTATAG	NNNNANNNN	GAGTTCTAAGAAGCTCCTTCTCA

TABLE 5
V segments targeted by each primer used for
the amplification of TCR β chain V segments.

SEQ	TCR b chain V
ID NO	segment name	Targeted V segment(s)

51	hTRBV1	hTRBV01
52	hTRBV2	hTRBV02
53	hTRBV3-1	hTRBV03-1, hTRBV03-2
54	hTRBV4-1	hTRBV04-1
55	hTRBV4-2	hTRBV04-2, hTRBV04-3
56	hTRBV5-1	hTRBV05-1
57	hTRBV5-2	hTRBV05-2
58	hTRBV5-3	hTRBV05-3
59	hTRBV5-4	hTRBV05-4, hTRBV05-5, hTRBV05-6,
		hTRBV05-7, hTRBV05-8
60	hTRBV6-1	hTRBV06-1
61	hTRBV6-2	hTRBV06-2, hTRBV06-3
62	hTRBV6-4	hTRBV06-4
63	hTRBV6-5	hTRBV06-5
64	hTRBV6-6	hTRBV06-6, hTRBV06-9
65	hTRBV6-7	hTRBV06-7
66	hTRBV6-8	hTRBV06-8
67	hTRBV7-1	hTRBV07-1
68	hTRBV7-2	hTRBV07-2, hTRBV07-8
69	hTRBV7-3	hTRBV07-3, hTRBV07-4
70	hTRBV7-5	hTRBV07-5
71	hTRBV7-6	hTRBV07-6, hTRBV07-7
72	hTRBV7-9	hTRBV07-9
73	hTRBV8-1	hTRBV08-1
74	hTRBV8-2	hTRBV08-2
75	hTRBV9	hTRBV09
76	hTRBV10-1	hTRBV10-1
77	hTRBV10-2	hTRBV10-2
78	hTRBV10-3	hTRBV10-3
79	hTRBV11-1	hTRBV11-1, hTRBV11-2, hTRBV11-3
80	hTRBV12-1	hTRBV12-1
81	hTRBV12-2	hTRBV12-2
82	hTRBV12-3	hTRBV12-3, hTRBV12-4, hTRBV12-5
83	hTRBV13	hTRBV13
84	hTRBV14	hTRBV14
85	hTRBV15	hTRBV15
86	hTRBV16	hTRBV16
87	hTRBV17	hTRBV17
88	hTRBV18	hTRBV18
89	hTRBV19	hTRBV19
90	hTRBV20	hTRBV20
91	hTRBV21	hTRBV21
92	hTRBV22	hTRBV22
93	hTRBV23	hTRBV23
94	hTRBV24	hTRBV24
95	hTRBV25	hTRBV25
96	hTRBV26	hTRBV26
97	hTRBV27	hTRBV27
98	hTRBV28	hTRBV28
99	hTRBV29	hTRBV29
100	hTRBV30	hTRBV30

TABLE 6
Primers for TCR gene amplification. Primer pair for sequencing of
TCR α genes: SEQ ID NO 101 and 102. Primer pair for sequencing
of TCR β genes: SEQ ID NO 101 and 103.

SEQ ID NO	Primer name	Primer sequence	TCR chain
101	Forward primer T7 TRAV/TRBV	TGTAATACGACTCACTATAG	α and β
102	Reverse primer PCR 1 TRAV	GGCCACAGCACTGTTGCTCTTGAAG	α
103	Reverse primer PCR 1 TRABV	CCACTGTGCACCTCCTTCCCATTC	β

TABLE 7
Reverse primers for TCR gene amplification that
did not result in successful
amplification of PCR products.

SEQ ID NO	Primer sequence	TCR chain
104	TCGACCAGCTTGACATCACAGG	α
105	CAGATTTGTTGCTCCAGGCCACAG	α
106	TCTGTGATATACACATCAGAATC	α
107	GAATCAAAATCGGTGAATAGGCAG	α
108	GGCAGACAGACTTGTCACTGGATT	α
109	TAGGACACCGAGGTAAAGCCAC	β
110	CTGGGTGACGGGTTTGGCCCTAT	β
111	TTGACAGCGGAAGTGGTTGC	β
112	GGCTGCTCAGGCAGTATCTGGAGTC	β
113	GCCAGGCACACCAGTGTGGCCTTTT	β

TABLE 8
Primers for addition of Next Generation Sequencing adapters. The primer portion
corresponding to the ILLUMINA ® adapters (forward and reverse) is underlined in forward
and reverse primers shown below. Primer pair for sequencing of TCR α genes: SEQ ID NOS:
114 and 115. Primer pair for sequencing of TCR β genes: SEQ ID NOS: 114 and 116.

SEQ			TCR
ID NO	Primer name	Primer sequence	chain
114	Forward primer Illumina_T7	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC	α and β
	TRAV/TRBV	TCTTCCGATCTTGTAATACGACTCACTATAG
115	Reverse primer PCR 2 TRAC	CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC	α
		GTGTGCTCTTCCGATCCTCAGCTGGTACACGGCAGGGTCA
116	Reverse primer PCR 2 TRBC	CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC	β
		GTGTGCTCTTCCGATCAAACACAGCGACCTCGGGTGGGAAC

Example 2: Exemplary Protocol for the SEQTR Method Using Nextera Adapters

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting material and RNA extraction

• • To obtain sufficient amounts of RNA in the extraction, a minimum of 500,000 T-cells were used as starting material. Alternatively, and especially in instances where fewer T-cells were available, T-cells were mixed with 50,000 mouse 3T3 cells that served as carrier. T-cell RNA was extracted using the RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's instruction with the following modification: Elution was performed with 20 μl of water preheated to 50° C. RNA quality and quantity was verified using a fragment analyzer.

2) cRNA synthesis by in vitro transcription (IVT):

• • In vitro transcription of isolated RNA was performed using the MessageAmp™ II aRNA Amplification Kit from Ambion® (Thermo Fisher Scientific), which contains enzymes, buffers and nucleotides required to perform the first and second strand cDNA and the in vitro transcription. The kit also provides all columns and reagents needed for the cDNA and cRNA purifications. RNA amplification was performed according to the manufacturer's instructions with the following modifications:
  • 1) Between 0.5 and 1 μg of total RNA as was used as starting material. 2) The IVT was performed in a final volume of 40 μl, and incubated at 37° C. for 16 h. Purified cRNA was quantified by absorbance using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific).

3) cDNA synthesis by reverse transcription:

• • The reverse transcription of the cRNA was performed with the SuperScript® III from Invitrogen (Thermo Fisher Scientific). The kit provides the enzyme, the buffer and the dithiothreitol (DTT) needed for the reaction. Deoxynucleotides (dNTPs) and RNAsin® Ribonuclease inhibitor were purchased from Promega. The sequences for the primers used for the reverse transcription can be found in Table 9 (primers for sequencing TCR α chain genes) and Table 10 (primers for sequencing TCR β chain genes).
  • 500 ng of cRNA were used as starting material for the reverse transcription. cRNA was mixed with 1 μl hTRAV or hTRBV primers mix (2 μM each) and 1 μl dNTP (25 mM) in a final volume of 13 μl. The mix was first incubated at 70° C. for 10 min, then at 50° C. for 30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μl SuperScript III and 1 μl RNAsin® were added to the mix. The samples were subsequently incubated for at 55° C. 1 h and then at 85° C. for 5 min. After the cDNA synthesis, 1 μg DNase-free RNase (Roche) was added to the cDNA and incubated at 37° C. for 30 min to remove the cRNA.

4) TCR gene amplification:

• • TCR gene amplification was performed using a Phusion® High-Fidelity DNA polymerase (New England Biolabs) under the following conditions:
  • PCR mix: 1 μl cDNA from step 3, 0.4 μl dNTPs (10 mM), 0.4 μl primer mix (20 μM Nextera 5′, 10 μM Reverse primer PCR 1 TRAV or 2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume of 10 μl.
  • PCR conditions:
    • 94° C. for 5 min
    • 20 cycles of
      • 98° C. for 10 s
      • 55° C. for 30 s
      • 72° C. for 30 s
    • 72° C. for 2 min
  • PCR products were purified using 1 μl of ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) according to the manufacturer's instructions.

5) Addition of Next Generation Sequencing adapters:

• • ILLUMINA® sequencing adapters were added by PCR using a Phusion® High-Fidelity DNA polymerase (New England Biolabs). The following mix was added to the 11 μl of PCR1: 1 μl dNTPs (10 mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl 5× buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.
  • PCR conditions:
    • 94° C. for 5 min
    • perform 25 cycles of:
      • 98° C. for 10 s
      • 55° C. for 30 s
      • 72° C. for 30 s
    • 72° C. for 2 min

6) TCR library purification:

• • 10 μl of the PCR product from step 5 were purified using an AMPURE XP beads (Beckman Coulter) according to the manufacturer's instruction. Samples could then directly be used for ILLUMINA® sequencing.

TABLE 9
Preferred primer sequences for amplification of TCR α chain V segments. N can be any
nucleotide. The sequences for primers presented in this table consist of three parts (listed
from 5′ to 3′): T7 adapter, barcode and TCR α chain V segment.

			Sequence
SEQ	Primer name	Sequence Nextera adapter	barcode portion	Sequence TCR α chain V segment
ID NO		portion of the primer	of the primer	portion of the primer

261	hTRAV1-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTTCTACAGGAGCTCCAGATGAAAG
		GTATAAGAGACAG
262	hTRAV1-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTTTTGAAGGAGCTCCAGATGAAAG
		GTATAAGAGACAG
263	hTRAV2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TGCTCATCCTCCAGGTGCGGGA
		GTATAAGAGACAG
264	hTRAV3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAAGAAACCATCTGCCCTTGTGA
		GTATAAGAGACAG
265	hTRAV4	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCTGCCCCGGGTTTCCCTGAGCGAC
		GTATAAGAGACAG
266	hTRAV5	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCTCTGCGCATTGCAGACACCCA
		GTATAAGAGACAG
267	hTRAV6	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTGTTTCATATCACAGCCTCCCA
		GTATAAGAGACAG
268	hTRAV7	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GCTTGTACATTACAGCCGTGCA
		GTATAAGAGACAG
269	hTRAV8-1/8-3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ATCTGAGGAAACCCTCTGTGCA
		GTATAAGAGACAG
270	hTRAV8-2/8-4	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ACCTGACGAAACCCTCAGCCCAT
		GTATAAGAGACAG
271	hTRAV8-5	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCTATGCCTGTCTTTACTTTAATC
		GTATAAGAGACAG
272	hTRAV8-6	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTTGAGGAAACCCTCAGTCCATAT
		GTATAAGAGACAG
273	hTRAV8-7	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAAACCATCAACCCATGTGAGTGA
		GTATAAGAGACAG
274	hTRAV9-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ACTTGGAGAAAGACTCAGTTCAA
		GTATAAGAGACAG
275	hTRAV9-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ACTTGGAGAAAGGCTCAGTTCAA
		GTATAAGAGACAG
276	hTRAV10	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTGCACATCACAGCCTCCCA
		GTATAAGAGACAG
277	hTRAV11	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTTTGGAATATCGCAGCCTCTCAT
		GTATAAGAGACAG
278	hTRAV12-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCCTGCTCATCAGAGACTCCAAG
		GTATAAGAGACAG
279	hTRAV12-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTCTGCTCATCAGAGACTCCCAG
		GTATAAGAGACAG
280	hTRAV12-3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCTTGTTCATCAGAGACTCACAG
		GTATAAGAGACAG
281	hTRAV13-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCTGCACATCACAGAGACCCAA
		GTATAAGAGACAG
282	hTRAV13-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCTCTGCAAATTGCAGCTACTCAA
		GTATAAGAGACAG
283	hTRAV14	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTGTCATCTCCGCTTCACAACTGG
		GTATAAGAGACAG
284	hTRAV15	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTTTTGAATATGCTGGTCTCTCAT
		GTATAAGAGACAG
285	hTRAV16	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCTGAAGAAACCATTTGCTCAAGA
		GTATAAGAGACAG
286	hTRAV17	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCTTGTTGATCACGGCTTCCCGG
		GTATAAGAGACAG
287	hTRAV18	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ACCTGGAGAAGCCCTCGGTGCA
		GTATAAGAGACAG
288	hTRAV19	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CACCATCACAGCCTCACAAGTCGT
		GTATAAGAGACAG
289	hTRAV20	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTTCTGCACATCACAGCCCCTA
		GTATAAGAGACAG
290	hTRAV21	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTTTATACATTGCAGCTTCTCAGCC
		GTATAAGAGACAG
291	hTRAV22	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTACATTTCCTCTTCCCAGACCAC
		GTATAAGAGACAG
292	hTRAV23	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CATTGCATATCATGGATTCCCAGC
		GTATAAGAGACAG
293	hTRAV24	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GCTATTTGTACATCAAAGGATCCC
		GTATAAGAGACAG
294	hTRAV25	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CAGCTCCCTGCACATCACAGCCA
		GTATAAGAGACAG
295	hTRAV26-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTGATCCTGCCCCACGCTACGCTGA
		GTATAAGAGACAG
296	hTRAV26-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTGATCCTGCACCGTGCTACCTTGA
		GTATAAGAGACAG
297	hTRAV27	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTTCTCTCCACATCACTGCAGCC
		GTATAAGAGACAG
298	hTRAV28	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GCCACCTATACATCAGATTCCCA
		GTATAAGAGACAG
299	hTRAV29	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCTCTGCACATTGTGCCCTCCCA
		GTATAAGAGACAG
300	hTRAV30	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCCTGTACCTTACGGCCTCCCAGCT
		GTATAAGAGACAG
301	hTRAV31	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTTATCATATCATCATCACAGCCA
		GTATAAGAGACAG
302	hTRAV32	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCTGCATATTACAGCCACCCAA
		GTATAAGAGACAG
303	hTRAV33	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ACCTCACCATCAATTCCTTAAAAC
		GTATAAGAGACAG
304	hTRAV34	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCTGCATATCACAGCCTCCCAG
		GTATAAGAGACAG
305	hTRAV35	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTTCCTGAATATCTCAGCATCCAT
		GTATAAGAGACAG
306	hTRAV36	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCTGAACATCACAGCCACCCAG
		GTATAAGAGACAG
307	hTRAV37	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCTGCACATACAGGATTCCCAG
		GTATAAGAGACAG
308	hTRAV38	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CAAGATCTCAGACTCACAGCTGG
		GTATAAGAGACAG
309	hTRAV39	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCGTCTCAGCACCCTCCACATCA
		GTATAAGAGACAG
310	hTRAV40	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCATTGTGAAATATTCAGTCCAGG
		GTATAAGAGACAG

TABLE 10
Preferred primer sequences for amplification of TCR β chain V segments. N can be
any nucleotide. The sequences for primers presented in this table consist of three parts
(listed from 5′ to 3′): T7 adapter, barcode and TCR β chain V segment.

			Sequence
SEQ		Sequence T7 adapter	barcode portion	Sequence TCR β chain V segment
ID NO	Primer name	portion of the primer	of the primer	portion of the primer

311	hTRBV1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTGGTCGCACTGCAGCAAGAAGA
		GTATAAGAGACAG
312	hTRBV2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GATCCGGTCCACAAAGCTGGAGGA
		GTATAAGAGACAG
313	hTRBV3-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CATCAATTCCCTGGAGCTTGGTGA
		GTATAAGAGACAG
314	hTRBV4-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTCACCTACACGCCCTGCAGCCAG
		GTATAAGAGACAG
315	hTRBV4-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TTCACCTACACACCCTGCAGCCAG
		GTATAAGAGACAG
316	hTRBV5-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAATGTGAGCACCTTGGAGCTGG
		GTATAAGAGACAG
317	hTRBV5-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TACTGAGTCAAACACGGAGCTAGG
		GTATAAGAGACAG
318	hTRBV5-3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GCTCTGAGATGAATGTGAGTGCCT
		GTATAAGAGACAG
319	hTRBV5-4	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTGAGCTGAATGTGAACGCCTT
		GTATAAGAGACAG
320	hTRBV6-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGTTCTCGCTCAGGCTGGAGT
		GTATAAGAGACAG
321	hTRBV6-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTGGGGTTGGAGTCGGCTGCTC
		GTATAAGAGACAG
322	hTRBV6-4	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCCCTCACGTTGGCGTCTGCTG
		GTATAAGAGACAG
323	hTRBV6-5	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCGCTCAGGCTGCTGTCGGC
		GTATAAGAGACAG
324	hTRBV6-6	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GATTTCCCGCTCAGGCTGGAGT
		GTATAAGAGACAG
325	hTRBV6-7	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCCCTCAAGCTGGAGTCAGCT
		GTATAAGAGACAG
326	hTRBV6-8	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCACTCAGGCTGGTGTCGGC
		GTATAAGAGACAG
327	hTRBV7-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTCTGAAGTTCCAGCGCACACA
		GTATAAGAGACAG
328	hTRBV7-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GATCCAGCGCACACAGCAGGAG
		GTATAAGAGACAG
329	hTRBV7-3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	ACTCTGAAGATCCAGCGCACAGA
		GTATAAGAGACAG
330	hTRBV7-5	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	AGATCCAGCGCACAGAGCAAGG
		GTATAAGAGACAG
331	hTRBV7-6	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CAGCGCACAGAGCAGCGGGACT
		GTATAAGAGACAG
332	hTRBV7-9	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGATCCAGCGCACAGAGCAGG
		GTATAAGAGACAG
333	hTRBV8-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCCTCAACCCTGGAGTCTACTA
		GTATAAGAGACAG
334	hTRBV8-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCCCAATCCTGGCATCCACCA
		GTATAAGAGACAG
335	hTRBV9	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTAAACCTGAGCTCTCTGGAGCT
		GTATAAGAGACAG
336	hTRBV10-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCCTCACTCTGGAGTCTGCTGC
		GTATAAGAGACAG
337	hTRBV10-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCCTCACTCTGGAGTCAGCTAC
		GTATAAGAGACAG
338	hTRBV10-3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TCCTCACTCTGGAGTCCGCTAC
		GTATAAGAGACAG
339	hTRBV11-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCACTCTCAAGATCCAGCCTGCA
		GTATAAGAGACAG
340	hTRBV12-1	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGGATCCAGCCCATGGAACCCA
		GTATAAGAGACAG
341	hTRBV12-2	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTGAAGATCCAGCCTGCAGAGC
		GTATAAGAGACAG
342	hTRBV12-3	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CAGCCCTCAGAACCCAGGGACT
		GTATAAGAGACAG
343	hTRBV13	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGCTCCTTGGAGCTGGGGGACT
		GTATAAGAGACAG
344	hTRBV14	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GGTGCAGCCTGCAGAACTGGAG
		GTATAAGAGACAG
345	hTRBV15	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GACATCCGCTCACCAGGCCTGG
		GTATAAGAGACAG
346	hTRBV16	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	TGAGATCCAGGCTACGAAGCTT
		GTATAAGAGACAG
347	hTRBV17	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAAGATCCATCCCGCAGAGCCG
		GTATAAGAGACAG
348	hTRBV18	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GGATCCAGCAGGTAGTGCGAGG
		GTATAAGAGACAG
349	hTRBV19	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CACTGTGACATCGGCCCAAAAG
		GTATAAGAGACAG
350	hTRBV20	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CTGACAGTGACCAGTGCCCATC
		GTATAAGAGACAG
351	hTRBV21	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGATCCAGTCCACGGAGTCAG
		GTATAAGAGACAG
352	hTRBV22	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTGAAGTTGGCCCACACCAGCCA
		GTATAAGAGACAG
353	hTRBV23	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	CCTGGCAATCCTGTCCTCAGAA
		GTATAAGAGACAG
354	hTRBV24	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGTCTGCCATCCCCAACCAGA
		GTATAAGAGACAG
355	hTRBV25	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GGAGTCTGCCAGGCCCTCACA
		GTATAAGAGACAG
356	hTRBV26	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAAGTCTGCCAGCACCAACCAG
		GTATAAGAGACAG
357	hTRBV27	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GGAGTCGCCCAGCCCCAACCAG
		GTATAAGAGACAG
358	hTRBV28	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GGAGTCCGCCAGCACCAACCAG
		GTATAAGAGACAG
359	hTRBV29	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GTGAGCAACATGAGCCCTGAAGA
		GTATAAGAGACAG
360	hTRBV30	TCGTCGGCAGCGTCAGATGT	HHHHHNNNN	GAGTTCTAAGAAGCTCCTTCTCA
		GTATAAGAGACAG

TABLE 11
Primers for TCR gene amplification. Primer pair for sequencing of TCR α genes:
SEQ ID NOs: 256 and 257. Primer pair for sequencing of TCR β genes: SEQ ID NOs: 256
and 258. The primer portion corresponding to the ILLUMINA ® adapters (forward and
reverse) is underlined in reverse primers shown below.

SEQ ID NO	Primer name	Primer sequence	TCR chain
256	Forward primer Nextera 5′	TCGTCGGCAGCGTC	α and β
257	Reverse primer PCR 1 TRAV	GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG	α
		GAATCAAAATCGGTGAATAGGCAG
258	Reverse primer PCR 1 TRBV	GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG	β
		GCCAGGCACACCAGTGTGGCCTTTT

TABLE 12
Primers used to add the full Nextera sequence to both TCRα and TCRβ.

SEQ ID NO	Primer name	Primer sequence	TCR chain
259	Index Read 1	CAAGCAGAAGACGGCATACGAGAT [i7] GTCTCGTGGGCTC	α and β
260	Index Read 2	AATGATACGGCGACCACCGAGATCTACAC [i5] TCGTCGGCAGCG	α

Example 3: Exemplary Protocol for the SEQTR Method without In Vitro Transcription

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting material and RNA extraction

• • To obtain sufficient amounts of RNA in the extraction, a minimum of 500,000 T-cells were used as starting material. Alternatively, and especially in instances where fewer T-cells were available, T-cells were mixed with 50,000 mouse 3T3 cells that served as carrier. T-cell RNA was extracted using the RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's instruction with the following modification: Elution was performed with 20 μl of water preheated to 50° C. RNA quality and quantity was verified using a fragment analyzer.

2) cDNA synthesis by reverse transcription:

• • The reverse transcription of the RNA was performed with the SuperScript® III from Invitrogen (Thermo Fisher Scientific) and oligo d(T). The kit provides the enzyme, the buffer and the dithiothreitol (DTT) needed for the reaction. Deoxynucleotides (dNTPs), oligo d(T) and RNAsin® Ribonuclease inhibitor were purchased from Promega.
  • 500 ng of RNA were used as starting material for the reverse transcription. RNA was mixed with 1 μl of oligo d(T) and 1 μl dNTP (25 mM) in a final volume of 13 μl. The mix was first incubated at 70° C. for 10 min, then at 50° C. for 30 s. 4 μl 5 × buffer, 1 μl DTT (100 mM), 1 μl SuperScript III and 1 μl RNAsin® were added to the mix. The samples were subsequently incubated for at 55° C. 1 h and then at 85° C. for 5 min.

3) Second strand cDNA synthesis:

• • cDNA was then used to synthesize the second strand, performed using the Phusion® High-Fidelity DNA polymerase (New England Biolabs) under the following conditions:
  • Mix: 20 μl cDNA from step 2, 4 μl dNTPs (10 mM), 2 μl TRAV primer mix (Table 9), 2 μl TRBV primers mix (Table 10), 20 μl 5× buffer, 1 μl Phusion® enzyme in a total volume of 100 μl.
  • Synthesis conditions:
    • 98° C. for 5 min
    • 40° C. for 30 s
    • 72° C. for 5 min

4) cDNA purification:

• • 100 μl of the cDNA product from step 3 were purified using an AMPURE XP beads (Beckman Coulter) according to the manufacturer's instruction.

5) TCR gene amplification:

• • TCR gene amplification was performed using a Phusion® High-Fidelity DNA polymerase (New England Biolabs) under the following conditions: PCR mix: 7 μl cDNA from step 4, 0.4 μl dNTPs (10 mM), 0.4 μl primer mix (20 μM Nextera5′, 10 μM Reverse primer PCR 1 TRAV or 2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume of 10 μl.
  • PCR conditions:
    • 94° C. for 5 min
    • 20 cycles of
      • 98° C. for 10 s
      • 55° C. for 30 s
      • 72° C. for 30 s
    • 72° C. for 2 min
  • PCR products were purified using 1 μl of ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) according to the manufacturer's instructions.

6) Addition of Next Generation Sequencing adapters:

• • ILLUMINA® sequencing adapters were added by PCR using a Phusion® High-Fidelity DNA polymerase (New England Biolabs). The following mix was added to the 11 μl of PCR1: 1 μl dNTPs (10 mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl 5× buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.
  • PCR conditions:
    • 94° C. for 5 min
    • perform 25 cycles of:
      • 98° C. for 10 s
      • 55° C. for 30 s
      • 72° C. for 30 s
    • 72° C. for 2 min

7) TCR library purification:

• • 10 μl of the PCR product from step 5 were purified using an AMPURE XP beads (Beckman Coulter) according to the manufacturer's instruction. Samples could then directly be used for ILLUMINA® sequencing.

Example 4: Sensitivity of the TCR Sequencing Method

One of the challenges of TCR sequencing are the small amounts of genetic material for each T-cell clone. In many cases, the number of T-cells that can be recovered from a given experiment is too small for researchers to directly extract sufficient amounts of RNA for a subsequent amplification of the TCR genes. In such instances, the T-cells of interest can be mixed with 3T3 mouse cells, which serve as a carrier.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to steps 2 to 4 of the SEQTR method outlined above (see Detailed Description of the Invention). PCR products were separated on an agarose gel and visualized.

No TCR-specific PCR products were observed in samples that only contained 3T3 cells (see FIG. 4). However, TCR-specific bands were detected in all other samples: Increasing amounts of CD8 positive T-cells in the samples were correlated with increasing amounts of TCR-specific PCR products and decreasing intensity of the unspecific dimer primer band. These data demonstrate that the SEQTR method is sensitive enough to amplify TCR genes from as little as 1,000 T-cells, with no detectable background signal from the 3T3 carrier cells.

Example 5: Specificity of the SEQTR Method

Another challenge of TCR sequencing is the lack of specific amplification of TCR genes from complex samples. Competing TCR sequencing technologies such as services offered by Adaptive Biotechnology are characterized by up to 90% unspecific amplification. As a result, only as little as 10% of all sequencing data are informative for TCR repertoire determination, increasing cost and duration of any project aiming to sequence TCR repertoires.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. TCR repertoires for the individual samples were sequenced using the SEQTR method, and the percentage of reads that corresponded to TCR or non-TCR sequences, respectively, was determined. As shown in FIG. 5, 93-97% of all sequencing reads indeed corresponded to TCR genes, independent of the amount of T-cells used as starting material. In summary, these data show that TCR amplification using the SEQTR method is highly specific even when as little as 1,000 T-cells are used as starting material.

Example 6: Unambiguous Identification of TCR Genes

In humans, the TCR locus comprises 54 different V segments for the TCR α chain and 65 different V segments for the TCR β chain. However, many of these V segments are highly homologous. Consequently, one of the big challenges of TCR sequencing is to successfully differentiate between two or more TCR gene segments with high degrees of homology. For instance, depending on the choice of primer used in the amplification of the TCR gene and the length of the generated PCR product, the resulting sequencing data might be compatible with more than one V or J segment (in other words, two or more TCR V or J segments show 100% homology in the sequenced region). In these cases, the TCR gene for a specific read cannot be unambiguously assigned/identified.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to the TCR sequencing method. Out of all the sequencing reads that were identified as TCR genes, it was assessed if the V or J segments could be identified unambiguously. The data show that between 95% and 97% of all TCR sequencing reads could be assigned to a specific TCR segment, even when using as little as 1,000 T-cells as genetic starting material (see FIG. 6). In summary, the data demonstrate the robustness of the SEQTR method as 90 to 93% of all reads can be used to identify TCR sequences once unspecific sequences and ambiguous TCR sequences have been removed.

Due to the homology between V segments, it can be sometimes difficult to clearly identify the TCR sequence. hTRBV6-2 and hTRBV6-3 cannot be differentiated as they have 100% homology and thus will code for the same TCR. Due to their sequences, hTRBV12-3 and hTRBV12-4 cannot be differentiated with the method disclosed herein. Only paired-end sequencing that will catch the 5′-end of the V segment can discriminate these two sequences. Thus the hTRBV12-3 and hTRBV12-4 were considered as a unique sequence for the analysis of the repertoire.

Example 7: Linearity of TCR Gene Amplification

Because non-linear amplification of individual TCR sequences can lead to an incorrect over- or underrepresentation of the affected TCR genes in the final TCR repertoire, linearity of amplification is a critical determinant of the reliability and quality of the TCR sequencing data.

To test linearity of TCR gene amplification in our system, a fixed amount of DNA encoding a known TCR sequence was diluted at different concentrations into a DNA pool representing a naïve CD8 repertoire. Subsequently, the TCR repertoire of each sample was analyzed with SEQTR.

The observed frequency of the known TCR sequence in the entire TCR repertoire was then sequenced for each dilution and compared to the expected frequency. The scatter plot in FIG. 7 shows an excellent correlation (R²=0.99) between the dilution and the frequency of the known TCR sequence in the repertoire observed after sequencing. These data confirm the linearity of the amplification and suggest that results obtained using the SEQTR technique are quantitative.

Example 8: Reproducibility of the SEQTR Method

The reproducibility of the method was tested by performing two independent technical replicates starting from the same sample. The frequencies for each V-J rearrangement in the TCR β chains were determined and compared between the two replicates, as illustrated in FIG. 8. Each sphere represents a single V-J rearrangement that was detected in both replicates. Each sphere represents a single V-J rearrangement with the size of a sphere indicating the relative frequency of the specific V-J recombination. Grey spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by less than two-fold. Black spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by more than two-fold. Consistent with common practice in the analysis of gene expression data, differences between replaces of less than 2-fold are not considered significant.

The data show that only 13% of all V-J rearrangements showed a significant frequency difference of more than two-fold between the two technical replicates (see FIG. 8 upper inset). However, as illustrated in FIG. 8, V-J recombinations that were significantly different between the technical replicates were rather poorly expressed, as indicated by the small sizes of the black spheres. Therefore, if the frequencies of the individual V-J rearrangement are taken into consideration, only 0.5% of the sequences showed more than a two-fold difference between the replicates (see FIG. 8 lower inset), demonstrating that the SEQTR method is very reproducible.

Example 9: Sequencing of Example Repertoires Using the SEQTR Method

The SEQTR method was tested on three different type of CD8 positive T-cells:

• • (1) T-cell population 1: CD8 positive T-cells isolated from peripheral blood mononuclear cells (PBMCs).
  • (2) T-cell population 2: CD8 positive T-cells as in population 1 were FACS sorted using tetramers. Tetramers are MHC molecules presenting a specific peptide, linked to fluorescent dye. Tetramers bind T-cell expressing a TCR that specifically recognizes the peptide. The fluorescent dye allows sorting of the the desired T-cells by FACS.
  • (3) T-cell population 3: CD8 positive T-cells as in population 2 that were subsequently expanded in vitro.

The relative frequencies of each V-J rearrangement were determined using the SEQTR method (see FIG. 9). As expected, the naïve TCR repertoire derived from PBMC (population 1) is highly diverse (see FIG. 9A). Almost all the possible V-J rearrangements are represented in the sample, with no single V-J rearrangement exhibiting a frequency of over 11% The repertoire of the tetramer sorted CD8 positive T-cell subset 2 (see FIG. 9B) is less diverse as compared to the naïve one. Not only are fewer V-J rearrangements present in the repertoire overall. Moreover, two V-J rearrangements are clearly dominant, exhibiting frequencies of over 20%. These V-J rearrangements represent the few T-cells that recognize the epitope TEDYMIHII (SEQ ID NO: 236) conjugated to the tetramer and that were enriched during the tetramer purification step. Finally, the rapid clonal expansion (population 3) of the tetramer-purified T-cells enhances the bias of the TCR repertoire towards the T-cell clones already dominating subset 2. Consequently, part of the low frequency V-J rearrangements are lost and not detected anymore (see FIG. 9C). In summary, these data illustrate that the SEQTR method is well suited to differentiate between TCR repertoires with different degrees of diversities.

Example 10: Comparison of the SEQTR Method with Low-Throughput Single Cell Cloning

In order to determine how accurate the TCR repertoire data obtained using the SEQTR method were as compared to the true TCR repertoire present in a given T-cell population, we compared our results to data obtained by single cell sequencing.

Tetramer-specific CD8 were sorted from PBMC by FACS. The recovered cell population was split in two. Half of the cells were subjected to the SEQTR method to sequence the TCR repertoire. For the other half of the cells, individual T-cell clones were isolated and expanded in vitro (single cell cloning). Once the clones were established, the TCR genes of each T-cell clones were amplified and sequenced using classical Sanger sequencing (see FIG. 10A).

Among the 42 individual clones tested using the single cell method, six different TCRs were identified (see FIG. 10B). Using the SEQTR method, 116 different TCR genes were found (the eight most frequently observed V-J rearrangements are shown in FIG. 10C, also see Table 13 and Table 14). Indeed, the five TCRs most frequently observed with the single cell cloning technique also correspond to the five clones most frequently observed when applying the SEQTR method. Overall, all six TCR clones identified with single cell sequencing are represented among the eight TCRs with the highest frequencies observed in the SEQTR method. In summary, these data suggest that the SEQTR method produces a true representation of the actual TCR repertoire of a given T-cell population.

TABLE 13
CDR3 regions of TCR clones identified using
the single cell sequencing method.

SEQ ID NO	CDR3 region
237	CASSRHVGGVPEAFFG
238	CASSIGRGSEQYFG
239	CASSDVLSGEAFFG
240	CASQGHKNTEAFFG
241	CASSLGPGGVKTNEKLFFG
242	CASSLGPGGVKTNEKLFFG

TABLE 14
Eight most frequently observed V-J
rearrangements of the 116 different TCR genes
identified using the SEQTR method.

SEQ ID NO	CDR3 region
243	CASSDVLSGEAFFG
244	CASQGHKNTEAFFG
245	CASSLGPGGVKTNEKLFFG
246	CASSIGRGSEQYFG
247	CASSRHVGGVPEAFFG
248	CASSASKGQPQHFG
249	CASQGHKNTEAFFG
250	CASSLGPGGVKTNEKLFFG

Example 11: TCR Sequencing Services Offered by Adaptive Biotechnology Provide Sequencing Data that May Reflect Up to 90% Unspecific TCR Amplification

Tumor samples from 16 patients were collected and the tumor cells were separated from the surrounding tissue (stroma). In addition, epitope-specific TIL were sorted by FACS from the tumor samples using tetramer staining (TET). Finally, the tumor cells were engrafted into humanized mice. After some time, the tumor was collected and epitope specific TIL were sorted by FACS.

DNA extraction was performed for each sample. DNA was sent to Adaptive Biotechnology for TCR sequencing (immunoSEQ® method, survey protocol 200,000-300,000 reads per sample).

In 80% of the samples, the immunoSEQ® method failed to generate 200,000 reads per samples, suggesting that the immunoSEQ® method fails to generate TCR repertoires with significant reliability.

Example 12: Amplification of TCR Genes from PBMC and CD4 Positive T-Cells

RNA was isolated from 10{circumflex over ( )}6 PBMC or 10{circumflex over ( )}6 CD4 positive T-cells, respectively, from three independent samples, The RNA was then subjected to steps 2 to 4 of the SEQTR method outlined above (see Detailed Description of the Invention). PCR products were separated on an agarose gel and visualized (see FIG. 12). Only TCR-specific bands are observed, suggesting that the SEQTR method cannot only be used for CD8 positive T-cells (see Example 7), but also for CD4 positive T-cells and even for T-cells that are part of a complex mixture of other PBMCs.

The foregoing examples and description of the embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference herein in their entireties.

As described and claimed herein, including in the accompanying drawings, reference is made to particular features, including method steps. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments, and in the disclosed methods, systems and kits generally.

Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

TABLE 15
TCR α chain V segments and binding sites for primers presented in Table 2 and
Table 9. The sequence for each V segment presented in this table consists
of three parts (listed from 5′ to 3′): Sequence upstream of primer
binding site, sequence of the primer
binding site, sequence downstream of the primer binding site.

					hTRAV
				Primer	sequence
				binding	downstream
V				site	of primer
segment	Primer	SEQ		within	binding
name	name	ID NO	hTRAV sequence upstream of primer binding site	hTRAV	site

hTRA	hTRA	117	ATGTGGGGAGCTTTCCTTCTCTATGTTTCCATGAAGATGGGAGGCACT	CTTCT	ACTCTGC
V01-1	V01-1		GCAGGACAAAGCCTTGAGCAGCCCTCTGAAGTGACAGCTGTGGAAGGA	ACAGG	CTCTTAC
			GCCATTGTCCAGATAAACTGCACGTACCAGACATCTGGGTTTTATGGG	AGCTC	TTCTGCG
			CTGTCCTGGTACCAGCAACATGATGGCGGAGCACCCACATTTCTTTCT	CAGAT	CTGTGAG
			TACAATGCTCTGGATGGTTTGGAGGAGACAGGTCGTTTTTCTTCATTC	GAAAG	AGA
			CTTAGTCGCTCTGATAGTTATGGTTACCTC
hTRA	hTRA	118	ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACT	CTTTT	ACTCTGC
V01-2	V0 1-2		ACAGGACAAAACATTGACCAGCCCACTGAGATGACAGCTACGGAAGGT	GAAGG	CTCTTAC
			GCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGG	AGCTC	CTCTGTG
			CTGTTCTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCT	CAGAT	CTGTGAG
			TACAATGTTCTGGATGGTTTGGAGGAGAAAGGTCGTTTTTCTTCATTC	GAAAG	AGA
			CTTAGTCGGTCTAAAGGGTACAGTTACCTC
hTRA	hTRA	119	ATGGCTTTGCAGAGCACTCTGGGGGCGGTGTGGCTAGGGCTTCTCCTC	TGCTC	GGCAGAT
V02	V02		AACTCTCTCTGGAAGGTTGCAGAAAGCAAGGACCAAGTGTTTCAGCCT	ATCCT	GCTGCTG
			TCCACAGTGGCATCTTCAGAGGGAGCTGTGGTGGAAATCTTCTGTAAT	CCAGG	TTTACTA
			CACTCTGTGTCCAATGCTTACAACTTCTTCTGGTACCTTCACTTCCCG	TGCGG	CTGTGCT
			GGATGTGCACCAAGACTCCTTGTTAAAGGCTCAAAGCCTTCTCAGCAG	GA	GTGGAGG
			GGACGATACAACATGACCTATGAACGGTTCTCTTCATCGC		A
hTRA	hTRA	120	ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCACATTGAGT	GAAGA	GCGACTC
V03	V03		GGGCTGAGAGCTCAGTCAGTGGCTCAGCCGGAAGATCAGGTCAACGTT	AACCA	CGCTTTG
			GCTGAAGGGAATCCTCTGACTGTGAAATGCACCTATTCAGTCTCTGGA	TCTGC	TACTTCT
			AACCCTTATCTTTTTTGGTATGTTCAATACCCCAACCGAGGCCTCCAG	CCTTG	GTGCTGT
			TTCCTTCTGAAATACATCACAGGGGATAACCTGGTTAAAGGCAGCTAT	TGA	GAGAGAC
			GGCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCACCT		A
hTRA	hTRA	121	ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTG	CCTGC	ACTGCTG
V04	V04		AGCCTTGCTAAGACCACCCAGCCCATCTCCATGGACTCATATGAAGGA	CCCGG	TGTACTA
			CAAGAAGTGAACATAACCTGTAGCCACAACAACATTGCTACAAATGAT	GTTTC	CTGCCTC
			TATATCACGTGGTACCAACAGTTTCCCAGCCAAGGACCACGATTTATT	CCTGA	GTGGGTG
			ATTCAAGGATACAAGACAAAAGTTACAAACGAAGTGGCCTCCCTGTTT	GCGAC	ACA
			ATCCCTGCCGACAGAAAGTCCAGCACTCTGAG
hTRA	hTRA	122	ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTG	TCTCT	GACTGGG
V05	V05		GACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGT	GCGCA	GACTCAG
			GTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGC	TTGCA	CTATCTA
			TCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTC	GACAC	CTTCTGT
			CAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAAGACCAA	CCA	GCAGAGA
			AGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTG		GTA
hTRA	hTRA	123	ATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTG	TTGTT	GCCTGCA
V06	V06		GACTGGGTGAAGAGCCAAAAGATAGAACAGAATTCCGAGGCCCTGAAC	TCATA	GACTCAG
			ATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTAT	TCACA	CTACCTA
			TCCCCAGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCT	GCCTC	CCTCTGT
			GTTTTCTTGCTACTCATACGTGAAAATGAGAAAGAAAAAAGGAAAGAA	CCA	GCTCTAG
			AGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGT		ACA
hTRA	hTRA	124	ATGGAGAAGATGCGGAGACCTGTCCTAATTATATTTTGTCTATGTCTT	GCTTG	GCCTGAA
V07	V07		GGCTGGGCAAATGGAGAAAACCAGGTGGAGCACAGCCCTCATTTTCTG	TACAT	GATTCAG
			GGACCCCAGCAGGGAGACGTTGCCTCCATGAGCTGCACGTACTCTGTC	TACAG	CCACCTA
			AGTCGTTTTAACAATTTGCAGTGGTACAGGCAAAATACAGGGATGGGT	CCGTG	TTTCTGT
			CCCAAACACCTATTATCCATGTATTCAGCTGGATATGAGAAGCAGAAA	CA	GCTGTAG
			GGAAGACTAAATGCTACATTACTGAAGAATGGAAGCA		ATG
hTRA	hTRA	125	ATGCTCCTGTTGCTCATACCAGTGCTGGGGATGATTTTTGCCCTGAGA	ATCTG	GTGGAGT
V08-1	V08-		GATGCCAGAGCCCAGTCTGTGAGCCAGCATAACCACCACGTAATTCTC	AGGAA	GACACAG
	1/08-3		TCTGAAGCAGCCTCACTGGAGTTGGGATGCAACTATTCCTATGGTGGA	ACCCT	CTGAGTA
			ACTGTTAATCTCTTCTGGTATGTCCAGTACCCTGGTCAACACCTTCAG	CTGTG	CTTCTGT
			CTTCTCCTCAAGTACTTTTCAGGGGATCCACTGGTTAAAGGCATCAAG	CA	GCCGTGA
			GGCTTTGAGGCTGAATTTATAAAGAGTAAATTCTCCTTTA		ATGC
hTRA	hTRA	126	ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGA	ACCTG	ATGAGCG
V08-2	V08-		GGAACCAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGTC	ACGAA	ACGCGGC
	2/08-4		TCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTAT	ACCCT	TGAGTAC
			TCACCATCTCTCTTCTGGTATGTGCAACACCCCAACAAAGGACTCCAG	CAGCC	TTCTGTG
			CTTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAAC	CAT	TTGTGAG
			GGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC		TGA
hTRA	hTRA	127	ATGCTCCTGGAGCTTATCCCACTGCTGGGGATACATTTTGTCCTGAGA	ATCTG	TTGGAGT
V08-3	V08-		ACTGCCAGAGCCCAGTCAGTGACCCAGCCTGACATCCACATCACTGTC	AGGAA	GATGCTG
	1/08-3		TCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGGGGCA	ACCCT	CTGAGTA
			ACACCTTATCTCTTCTGGTATGTCCAGTCCCCCGGCCAAGGCCTCCAG	CTGTG	CTTCTGT
			CTGCTCCTGAAGTACTTTTCAGGAGACACTCTGGTTCAAGGCATTAAA	CA	GCTGTGG
			GGCTTTGAGGCTGAATTTAAGAGGAGTCAATCTTCCTTCA		GTGC
hTRA	hTRA	128	ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACCCTGGGA	ACCTG	ATGAGCG
V08-4	V08-		GGAACCAGAGCCCAGTCGGTGACCCAGCTTGGCAGCCACGTCTCTGTC	ACGAA	ACGCGGC
	2/08-4		TCTGAAGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTT	ACCCT	TGAGTAC
			CCACCATATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAG	CAGCC	TTCTGTG
			CTTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAAC	CAT	CTGTGAG
			GGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC		TGACACA
hTRA	hTRA	129	ATGCTCCTGGTGCTCATCCCACTGCTGGGGATACATTTTGTCCTGAGT	CCTAT	TCTTAAT
V08-5	V08-5		GAGAACTGTCAGAGCCCAGTCAGTGACCCAGCCTGACATCCGCATCAC	GCCTG	CCTGTCA
			TGTCTCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGG	TCTTT	GCTGAGG
			GGCGATGTTGTGGGAAGTCAGGGACCCCAAACGGAGGGACCGGCTGAA	ACTTT	AGGATGT
			GCCATGGCAGAAGAATGTGGATTGTGAAGATTTCATGGACATTTATTA	AATC	ATGTCAC
			GTTCCCCAAATTAATACTTTTATAATTTCTTATGCCTCTCTTTACTGC		C
			AATCTCTAAACATAAATTGTAAAGATTTCATGGACACTTATCACTTCC
			CCAATCAATACCCCTGTGATTT
hTRA	hTRA	130	ATGCTCCTGCTGCTCGTCCCAGCGTTCCAGGTGATTTTTACCCTGGGA	CTTGA	AAGCGAC
V08-6	V08-6		GGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTC	GGAAA	ACGGCTG
			TTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTT	CCCTC	AGTACTT
			TCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAG	AGTCC	CTGTGCT
			CTTCTCCTGAAGTATTTATCAGGATCCACCCTGGTTAAAGGCATCAAC	ATAT	GTGAGTG
			GGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCA		A
hTRA	hTRA	131	ATGCTCTTAGTGGTCATTCTGCTGCTTGGAATGTTCTTCACACTGAGA	GAAAC	TGCTGCT
V08-7	V08-7		ACCAGAACCCAGTCGGTGACCCAGCTTGATGGCCACATCACTGTCTCT	CATCA	GAGTACT
			GAAGAAGCCCCTCTGGAACTGAAGTGCAACTATTCCTATAGTGGAGTT	ACCCA	TCTGTGC
			CCTTCTCTCTTCTGGTATGTCCAATACTCTAGCCAAAGCCTCCAGCTT	TGTGA	TGTGGGT
			CTCCTCAAAGACCTAACAGAGGCCACCCAGGTTAAAGGCATCAGAGGT	GTGA	GACAGG
			TTTGAGGCTGAATTTAAGAAGAGCGAAACCTCCTTCTACCTGAG
hTRA	hTRA	132	ATGAATTCTTCTCCAGGACCAGCGATTGCACTATTCTTAATGTTTGGG	ACTTG	GAGTCAG
V09-1	V09-1		GGAATCAATGGAGATTCAGTGGTCCAGACAGAAGGCCAAGTGCTCCCC	GAGAA	ACTCCGC
			TCTGAAGGGGATTCCCTGATTGTGAACTGCTCCTATGAAACCACACAG	AGACT	TGTGTAC
			TACCCTTCCCTTTTTTGGTATGTCCAATATCCTGGAGAAGGTCCACAG	CAGTT	TTCTGTG
			CTCCACCTGAAAGCCATGAAGGCCAATGACAAGGGAAGGAACAAAGGT	CAA	CTCTGAG
			TTTGAAGCCATGTACCGTAAAGAAACCACTTCTTTCC		TGA
hTRA	hTRA	133	ATGAACTATTCTCCAGGCTTAGTATCTCTGATACTCTTACTGCTTGGA	ACTTG	GTGTCAG
V09-2	V09-2		AGAACCCGTGGAAATTCAGTGACCCAGATGGAAGGGCCAGTGACTCTC	GAGAA	ACTCAGC
			TCAGAAGAGGCCTTCCTGACTATAAACTGCACGTACACAGCCACAGGA	AGGCT	GGTGTAC
			TACCCTTCCCTTTTCTGGTATGTCCAATATCCTGGAGAAGGTCTACAG	CAGTT	TTCTGTG
			CTCCTCCTGAAAGCCACGAAGGCTGATGACAAGGGAAGCAACAAAGGT	CAA	CTCTGAG
			TTTGAAGCCACATACCGTAAAGAAACCACTTCTTTCC		TGA
hTRA	hTRA	134	ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTT	CTGCA	GCTCAGC
V10	V10		TATAGGGGGAATGGCAAAAACCAAGTGGAGCAGAGTCCTCAGTCCCTG	CATCA	GATTCAG
			ATCATCCTGGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTG	CAGCC	CCTCCTA
			AGCCCCTTCAGCAACTTAAGGTGGTATAAGCAAGATACTGGGAGAGGT	TCCCA	CATCTGT
			CCTGTTTCCCTGACAATCATGACTTTCAGTGAGAACACAAAGTCGAAC		GTGGTGA
			GGAAGATATACAGCAACTCTGGATGCAGACACAAAGCAAAGCTCT		GCG
hTRA	hTRA	135	ACGGAGAAGCCCTTGGGAGTTTCATTCTTGATTTCCTCCTGGCAGCTG	GTTTG	CTGGGAG
V11	VII		TGCTGGGTGAATAGACTACATACACTGGAGCAGAGTCCTTCATTCCTG	GAATA	ATTCAGC
			AATATTCAGGAGGGAATGCATGCCGTTCTTAATTGTACTTATCAGGAG	TCGCA	CACCTAC
			AGAACACTCTTCAATTTCCACTGGTTCCGGCAGGATCCGGGGAGAAGA	GCCTC	TTCTGTG
			CTTGTGTCTTTGACCTTAATTCAATCAAGCCAGAAGGAGCAGGGAGAC	TCAT	CTTTGC
			AAATATTTTAAAGAACTGCTTGGAAAAGAAAAATTTTATAGT
hTRA	hTRA	136	ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGC	CCCTG	CTCAGTG
V12-1	V12-1		TGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTC	CTCAT	ATTCAGC
			AATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAAC	CAGAG	CACCTAC
			AGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAA	ACTCC	CTCTGTG
			CCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAATGAAGATGGAAGG	AAG	TGGTGAA
			TTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTT		CA
hTRA	hTRA	137	ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGC	CTCTG	CCCAGTG
V12-2	V12-2		TGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTC	CTCAT	ATTCAGC
			AGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGAC	CAGAG	CACCTAC
			CGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGC	ACTCC	CTCTGTG
			CCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGA	CAG	CCGTGAA
			AGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTT
hTRA	hTRA	138	ATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGC	CCTTG	CCCAGTG
V12-3	V12-3		TGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGGATCCTGGACCACTC	TTCAT	ATTCAGC
			AGTGTTCCAGAGGGAGCCATTGTTTCTCTCAACTGCACTTACAGCAAC	CAGAG	CACCTAC
			AGTGCTTTTCAATACTTCATGTGGTACAGACAGTATTCCAGAAAAGGC	ACTCA	CTCTGTG
			CCTGAGTTGCTGATGTACACATACTCCAGTGGTAACAAAGAAGATGGA	CAG	CAATGAG
			AGGTTTACAGCACAGGTCGATAAATCCAGCAAGTATATCT		CGCACAG
hTRA	hTRA	139	ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGAC	TCCCT	CCTGAAG
V13-1	V13-1		TTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTC	GCACA	ACTCGGC
			CAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCC	TCACA	TGTCTAC
			TCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAGGACCTCAG	GAGAC	TTCTGTG
			CTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGA	CCAA	CAGCAAG
			ATTGCTGTTACATTGAACAAGACAGCCAAACATTTC		TA
hTRA	hTRA	140	ATGGCAGGCATTCGAGCTTTATTTATGTACTTGTGGCTGCAGCTGGAC	TCTCT	CCTGGAG
V13-2	V13-2		TGGGTGAGCAGAGGAGAGAGTGTGGGGCTGCATCTTCCTACCCTGAGT	GCAAA	ACTCAGC
			GTCCAGGAGGGTGACAACTCTATTATCAACTGTGCTTATTCAAACAGC	TTGCA	TGTCTAC
			GCCTCAGACTACTTCATTTGGTACAAGCAAGAATCTGGAAAAGGTCCT	GCTAC	TTTTGTG
			CAATTCATTATAGACATTCGTTCAAATATGGACAAAAGGCAAGGCCAA	TCAA	CAGAGAA
			AGAGTCACCGTTTTATTGAATAAGACAGTGAAACATCTC		TA
hTRA	hTRA	141	ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTA	TTGTC	GGGACTC
V14	V14		GGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTC	ATCTC	AGCAATG
			GTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGT	CGCTT	TATTTCT
			GATCAAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAA	CACAA	GTGCAAT
			ATGATTTTTCTTATTTATCAGGGGTCTTATGACGAGCAAAATGCAACA	CTGG	GAGAGAG
			GAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAAC		GG
			C
hTRA	hTRA	142	ATGTATACGTATGTAACAAACCTGCGCGTTGTGCACATGTACCCTAGA	GTTTT	CCTGGAG
V15	V15		ACGGGTGAACAGCCTCCATATTCTGGAGTAGAGTCCTTCATTCATTCC	GAATA	ATTCAGG
			TGAGTATCCGGGAGGGAATGCACAACATTCTTAATTGCACTTATGAGG	TGCTG	CACCTAC
			AGAGAACGTTCTCTTAACTTCTACTGGTTCTGGCAGGGTCTGGAAAAG	GTCTC	TTCTGTG
			GACTTGTGTCTTTGACCTTAATTCAATCAAGCCAGATGGAGGAGGGAG	TCAT	CTTTGAG
			ACAAACATTTTAAAGAAGCGCTTGGAAAAGAGAAGTTTTATAGT		G
hTRA	hTRA	143	ATGAAGCCCACCCTCATCTCAGTGCTTGTGATAATATTTATACTCAGA	CCTGA	GGAAGAC
V16	V16		GGAACAAGAGCCCAGAGAGTGACTCAGCCCGAGAAGCTCCTCTCTGTC	AGAAA	TCAGCCA
			TTTAAAGGGGCCCCAGTGGAGCTGAAGTGCAACTATTCCTATTCTGGG	CCATT	TGTATTA
			AGTCCTGAACTCTTCTGGTATGTCCAGTACTCCAGACAACGCCTCCAG	TGCTC	CTGTGCT
			TTACTCTTGAGACACATCTCTAGAGAGAGCATCAAAGGCTTCACTGCT	AAGA	CTAAGTG
			GACCTTAACAAAGGCGAGACATCTTTCCA		G
hTRA	hTRA	144	ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTG	TCCTT	GCAGCAG
V17	V17		GCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGC	GTTGA	ACACTGC
			ATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGT	TCACG	TTCTTAC
			ATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTC	GCTTC	TTCTGTG
			CACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGA	CCGG	CTACGGA
			TTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGT		CG
hTRA	hTRA	145	ATGCTGTCTGCTTCCTGCTCAGGACTTGTGATCTTGTTGATATTCAGA	ACCTG	GCTGTCG
V18	V18		AGGACCAGTGGAGACTCGGTTACCCAGACAGAAGGCCCAGTTACCCTC	GAGAA	GACTCTG
			CCTGAGAGGGCAGCTCTGACATTAAACTGCACTTATCAGTCCAGCTAT	GCCCT	CCGTGTA
			TCAACTTTTCTATTCTGGTATGTCCAGTATCTAAACAAAGAGCCTGAG	CGGTG	CTACTGC
			CTCCTCCTGAAAAGTTCAGAAAACCAGGAGACGGACAGCAGAGGTTTT	CA	GCTCTGA
			CAGGCCAGTCCTATCAAGAGTGACAGTTCCTTCC		GA
hTRA	hTRA	146	ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTT	CACCA	GGACTCA
V19	V19		GTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCT	TCACA	GCAGTAT
			GTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGT	GCCTC	ACTTCTG
			GATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAA	ACAAG	TGCTCTG
			TTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATA	TCGT	AGTGAGG
			AGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAAC		C
			TT
hTRA	hTRA	147	ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTT	TTTCT	AACCTGA
V20	V20		GGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTG	GCACA	AGACTCA
			AGACTCCAGGAGGGAGAGAGTAGCAGTCTTAACTGCAGTTACACAGTC	TCACA	GCCACTT
			AGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGC	GCCCC	ATCTCTG
			CCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAA	TA	TGCTGTG
			GAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGC		CAGG
hTRA	hTRA	148	ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGG	CTTTA	TGGTGAC
V21	V21		GTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTC	TACAT	TCAGCCA
			CCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCT	TGCAG	CCTACCT
			ATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACA	CTTCT	CTGTGCT
			TCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGA	CAGCC	GTGAGG
			CTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTA
hTRA	hTRA	149	ATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTT	GTACA	AGACTCA
V22	V22		TGCTGTGTGAGAGGAATACAAGTGGAGCAGAGTCCTCCAGACCTGATT	TTTCC	GGCGTTT
			CTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCT	TCTTC	ATTTCTG
			GTGAACAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATC
			AACCTGTTTTACATTCCCTCAGGGACAAAACAGAATGGAAGATTAAGC	CCAGA	TGCTGTG
			GCCACGACTGTCGCTACGGAACGCTACAGCTTATT	CCAC	GAGC
hTRA	hTRA	150	ATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTA	CATTG	CTGGAGA
V23	V23		TGCTGGGTGAGTGGCCAACAGAAGGAGAAAAGTGACCAGCAGCAGGTG	CATAT	CTCAGCC
			AAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATT	CATGG	ACCTACT
			ATAAACTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTAC	ATTCC	TCTGTGC
			CAACAATTCCCTGGGAAAGGCCCTGCATTATTGATAGCCATACGTCCA	CAGC	AGCAAGC
			GATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAA		A
			AGTGCCAAGCAGTTCT
hTRA	hTRA	151	ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCAT	GCTAT	AGCCTGA
V24	V24		CTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCA	TTGTA	AGACTCA
			CTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCT	CATCA	GCCACAT
			TCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAA	AAGGA	ACCTCTG
			AGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAG	TCCC	TGCCTTT
			AAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACA		A
hTRA	hTRA	152	ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAG	CAGCT	CCCAGAC
V25	V25		GTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAA	CCCTG	TACAGAT
			GAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGC	CACAT	GTAGGAA
			AATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTG	CACAG	CCTACTT
			ATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACA	CCA	CTGTGCA
			TTTCAGTTTGGAGAAGCAAAAAAGAA		GGG
hTRA	hTRA	153	ATGAGGCTGGTGGCAAGAGTAACTGTGTTTCTGACCTTTGGAACTATA	TTGAT	GAGACAC
V26-1	V26-1		ATTGATGCTAAGACCACCCAGCCCCCCTCCATGGATTGCGCTGAAGGA	CCTGC	TGCTGTG
			AGAGCTGCAAACCTGCCTTGTAATCACTCTACCATCAGTGGAAATGAG	CCCAC	TACTATT
			TATGTGTATTGGTATCGACAGATTCACTCCCAGGGGCCACAGTATATC	GCTAC	GCATCGT
			ATTCATGGTCTAAAAAACAATGAAACCAATGAAATGGCCTCTCTGATC	GCTGA	CAGAGTC
			ATCACAGAAGACAGAAAGTCCAGCACC		G
hTRA	hTRA	154	ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATG	TTGAT	GAGATGC
V26-2	V26-2		GGTGATGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAA	CCTGC	TGCTGTG
			GAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGAT	ACCGT	TACTACT
			TACATACATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTG	GCTAC	GCATCCT
			ATTCATGGTCTTACAAGCAATGTGAACAACAGAATGGCCTCTCTGGCA	CTTGA	GAGAGAC
			ATCGCTGAAGACAGAAAGTCCAGTACC
hTRA	hTRA	155	ATGGTCCTGAAATTCTCCGTGTCCATTCTTTGGATTCAGTTGGCATGG	GTTCT	CAGCCTG
V27	V27		GTGAGCACCCAGCTGCTGGAGCAGAGCCCTCAGTTTCTAAGCATCCAA	CTCCA	GTGATAC
			GAGGGAGAAAATCTCACTGTGTACTGCAACTCCTCAAGTGTTTTTTCC	CATCA	AGGCCTC
			AGCTTACAATGGTACAGACAGGAGCCTGGGGAAGGTCCTGTCCTCCTG	CTGCA	TACCTCT
			GTGACAGTAGTTACGGGTGGAGAAGTGAAGAAGCTGAAGAGACTAACC	GCC	GTGCAGG
			TTTCAGTTTGGTGATGCAAGAAAGGACA		AG
hTRA	hTRA	156	ATGAAGGCATTAATAGGAATCTTGCTGGGCTTCCTGTGGATACAGATT	GCCAC	GCCTGAG
V28	V28		TGCTCGCAAATGAAAGTGGAGCAGAGTCCTCAGGTCCTGATCCTCCAA	CTATA	GACTCAG
			GAGGGAAGAAATTCATTCCTGGTGTGCAGTTGTTCTATTTACATGATC	CATCA	CTATTTA
			CGTGTGCAGTGGTTTCATCAAAAGCCTGGAGGACCCCTCATGTCCTTA	GATTC	CTTCTGT
			TTTAACATTAATTCAGGAATACAGCAAAAAAGAAGACTAAAATCCGCA	CCA	GCTGTGG
			GTCAAAGCTGAGGAACTTTATG		GGA
hTRA	hTRA	157	ATGGCCATGCTCCTGGGGGCATCAGTGCTGATTCTGTGGCTTCAGCCA	TCTCT	GCCTGGA
V29	V29		GACTGGGTAAACAGTCAACAGAAGAATGATGACCAGCAAGTTAAGCAA	GCACA	GACTCTG
			AATTCACCATCCCTGAGCGTCCAGGAAGGAAGAATTTCTATTCTGAAC	TTGTG	CAGTGTA
			TGTGACTATACTAACAGCATGTTTGATTATTTCCTATGGTACAAAAAA	CCCTC	CTTCTGT
			TACCCTGCTGAAGGTCCTACATTCCTGATATCTATAAGTTCCATTAAG	CCA	GCAGCAA
			GATAAAAATGAAGATGGAAGATTCACTGTCTTCTTAAACAAAAGTGCC		GCG
			AAGCACCTC
hTRA	hTRA	158	ATGGAGACTCTCCTGAAAGTGCTTTCAGGCACCTTGTTGTGGCAGTTG	CCCTG	CAGTTAC
V30	V30		ACCTGGGTGAGAAGCCAACAACCAGTGCAGAGTCCTCAAGCCGTGATC	TACCT	TCAGGAA
			CTCCGAGAAGGGGAAGATGCTGTCATCAACTGCAGTTCCTCCAAGGCT	TACGG	CCTACTT
			TTATATTCTGTACACTGGTACAGGCAGAAGCATGGTGAAGCACCCGTC	CCTCC	CTGCGGC
			TTCCTGATGATATTACTGAAGGGTGGAGAACAGAAGGGTCATGAAAAA	CAGCT	ACAGAGA
			ATATCTGCTTCATTTAATGAAAAAAAGCAGCAAAGCT
hTRA	hTRA	159	ATGACTGTTGGCAGCATATTACGGGCACTCATGGCCTCTGCCTTCCTT	CTTAT	GAAGACC
V31	V31		GCATGTCACAGAGGGTCATTCAATCCCAACCAGCAATATCTACGCAGG	CATAT	TGCAACA
			AGGGTGAGACCGTGAAACTGGACTGTGCATACAAAACTAATATTGTAT	CATCA	TATTTCT
			ATTACATATTGTATTGGTACAAAAGGTCTCCCAATGGGAAGATTATTT	TCACA	GTTGTCT
			TCCTCATTTATCAGCAAACAGATGCAGAAACCAATGCGACACAGGGTC	GCCA	CAAAGAG
			AATATTCTGTGAGCTTCCAGAAAACAACTAAAACTATTCAG		CC
hTRA	hTRA	160	ATGGCAAGAAGAATGGAAAAGTCCCTGGGAGCTTTATTCAAATTCAGC	TCCCT	CCAGGAG
V32	V32		TGAAGCTGGCCAAGAAAAGGATGTGATACAGAGTTATTCAAATCTAAA	GCATA	ACTCATT
			TGTCTAGGAGAGAGAAATGGCCGTTATTAATGACAGTTATACAGATGG	TTACA	CCTGTAC
			AGCTTTGAATTATTTCTGTTGGTACAAGAAGAAAACGGGGAAGGCCCT	GCCAC	TTCTGTG
			AATATCTTAATGGAGATTCATTCAAATGTGGATAGAAAACAGGACAGA	CCAA	CAGTGAG
			AGGCTCACTGTACTGTTGAATAAAAATGCTAAACATGTC		AACACA
hTRA	hTRA	161	ATGCTCTGCCCTGGCCTGCTGTGGGCATTCGTGGTCCCCTTTGGCTTC	ACCTC	TGACTCA
V33	V33		AGATCCAGCATGGCTCAGAAAGTAACCCAAGTTCAGACCACAGTAACT	ACCAT	GCCAAGT
			AGGCAGAAAGGAGTAGCTGTGACCTTGGACTGCATGTTTGAAACCAGA	CAATT	ACTTCTG
			TAGAATTCGTACACTTTATACTGGTACAAGCAACAAGCAACCTCCCAG	CCTTA	TGCTCTC
			TGAAGAGATGGTTTTCCTTATTCATCAGGGTTATTCTAAGTCAAATGC	AAAC	AGGAATC
			AAAGCCTGTGAACTTTGAAAAAAAGAAAAAGTTCATCA		C
hTRA	hTRA	162	ATGGAGACTGTTCTGCAAGTACTCCTAGGGATATTGGGGTTCCAAGCA	TCCCT	CCCAGCC
V34	V34		GCCTGGGTCAGTAGCCAAGAACTGGAGCAGAGTCCTCAGTCCTTGATC	GCATA	ATGCAGG
			GTCCAAGAGGGAAAGAATCTCACCATAAACTGCACGTCATCAAAGACG	TCACA	CATCTAC
			TTATATGGCTTATACTGGTATAAGCAAAAGTATGGTGAAGGTCTTATC	GCCTC	CTCTGTG
			TTCTTGATGATGCTACAGAAAGGTGGGGAAGAGAAAAGTCATGAAAAG	CCAG	GAGCAGA
			ATAACTGCCAAGTTGGATGAGAAAAAGCAGCAAAGT		CA
hTRA	hTRA	163	ATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGG	CTTCC	ACCTAGT
V35	V35		GTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAG	TGAAT	GATGTAG
			GAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAAC	ATCTC	GCATCTA
			ACCTGGCTATGGTACAAGCAGGAACCTGGGGAAGGTCCTGTCCTCTTG	AGCAT	CTTCTGT
			ATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACT	CCAT	GCTGGGC
			GCTCAGTTTGGTATAACCAGAAAGGACAG		AG
hTRA	hTRA	164	ATGATGAAGTGTCCACAGGCTTTACTAGCTATCTTTTGGCTTCTACTG	TCCTG	ACCGGAG
V36	V36		AGCTGGGTGAGCAGTGAAGACAAGGTGGTACAAAGCCCTCTATCTCTG	AACAT	ACTCGGC
			GTTGTCCACGAGGGAGACACCGTAACTCTCAATTGCAGTTATGAAGTG	CACAG	CATCTAC
			ACTAACTTTCGAAGCCTACTATGGTACAAGCAGGAAAAGAAAGCTCCC	CCACC	CTCTGTG
			ACATTTCTATTTATGCTAACTTCAAGTGGAATTGAAAAGAAGTCAGGA	CAG	CTGTGGA
			AGACTAAGTAGCATATTAGATAAGAAAGAACTTTCCAGCA		GG
hTRA	hTRA	165	ATGGAAACTCCACTGAGCACTCTGCTGCTGCTCCTCTGTGTGCAGCTG	TCCCT	CTCCATG
V37	V37		ACCTGGTCAAATGGACAACTGCCAGTGGAACAGAATGCTCCTTCCCTG	GCACA	ACTCAAC
			AAAGTCAAGGAAGGTGACAGCGTCACACTGAACTGCAGTTACAGAGAC	TACAG	CACATTC
			AGCCCTTCAGATTTCTTCAGTGGTTCAGGCAGGATCCTGAGGAAGGCC	GATTC	TTCTGCG
			TCATTTCCCTGATACAAATGCTATCAACTGTGAGAGAGAAGATCAGTG	CCAG	CAGCAAG
			GAAGATTCACAGCCAGGCTTAAAAAAGGAGACCAGCACATT		CA
hTRA	hTRA	166	ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTT	CAAGA	GGGACAC
V38-1	V38		GAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCT	TCTCA	TGCGATG
			GTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGT	GACTC	TATTTCT
			GAGAATAATTATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAG	ACAGC	GTGCTTT
			ATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACG	TGG	CATGAAG
			GAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGT		CA
			CT
hTRA	hTRA	167	ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTT	CAAGA	GGGATGC
V38-2	V38		GAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCT	TCTCA	CGCGATG
			GTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGT	GACTC	TATTTCT
			GAGAGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAG	ACAGC	GTGCTTA
			ATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACA	TGG	TAGGAGC
			GAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGT		G
			CT
hTRA	hTRA	168	ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTA	CCGTC	CAGCTGC
V39	V39		AGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATGCAG	TCAGC	CGTGCAT
			GAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGAC	ACCCT	GACCTCT
			AGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTG	CCACA	CTGCCAC
			TTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGACGATTAATG	TCA	CTACTTC
			GCCTCACTTGATACCAAAGC		TGTGCCG
					TGGACA
hTRA	hTRA	169	ATGAACTCCTCTCTGGACTTTCTAATTCTGATCTTAATGTTTGGAGGA	CCATT	TATCAGA
V40	V40		ACCAGCAGCAATTCAGTCAAGCAGACGGGCCAAATAACCGTCTCGGAG	GTGAA	CTCAGCC
			GGAGCATCTGTGACTATGAACTGCACATACACATCCACGGGGTACCCT	ATATT	GTGTACT
			ACCCTTTTCTGGTATGTGGAATACCCCAGCAAACCTCTGCAGCTTCTT	CAGTC	ACTGTCT
			CAGAGAGAGACAATGGAAAACAGCAAAAACTTCGGAGGCGGAAATATT	CAGG	TCTGGGA
			AAAGACAAAAACTCCC		GA
hTRA	hTRA	170	ATGGTGAAGATCCGGCAATTTTTGTTGGCTATTTTGTGGCTTCAGCTA	CTGCA	TCCCAGA
V41	V10		AGCTGTGTAAGTGCCGCCAAAAATGAAGTGGAGCAGAGTCCTCAGAAC	CATCA	GACTCTG
			CTGACTGCCCAGGAAGGAGAATTTATCACAATCAACTGCAGTTACTCG	CAGCC	CCGTCTA
			GTAGGAATAAGTGCCTTACACTGGCTGCAACAGCATCCAGGAGGAGGC	TCCCA	CATCTGT
			ATTGTTTCCTTGTTTATGCTGAGCTCAGGGAAGAAGAAGCATGGAAGA		GCTGTCA
			TTAATTGCCACAATAAACATACAGGAAAAGCACAGCTCC		GA

TABLE 16
TCR β chain V segments and binding sites for primers presented in Table 4 and
Table 10. The sequence for each V segments presented in this table
consists of three parts (listed from 5′ to 3′): Sequence
upstream of primer binding site, sequence of the primer
binding site and sequence downstream of the primer binding site.

					hTRBV
				Primer	sequence
				binding	downstream
V				site	of primer
segment	Primer	SEQ		within	binding
name	name	ID NO	hTRBV sequence upstream of primer binding site	hTRBV	site

hTRB	hTRB	171	ATGGGCTGAAGTCTCCACTGTGGTGTGGTCCATTGTCTCAGGCTCCAT	GTGGT	CTCAGCT
V01	V01		GGATACTGGAATTACCCAGACACCAAAATACCTGGTCACAGCAATGGG	CGCAC	GCGTATC
			GAGTAAAAGGACAATGAAACGTGAGCATCTGGGACATGATTCTATGTA	TGCAG	TCTGCAC
			TTGGTACAGACAGAAAGCTAAGAAATCCCTGGAGTTCATGTTTTACTA	CAAGA	CAGCAGC
			CAACTGTAAGGAATTCATTGAAAACAAGACTGTGCCAAATCACTTCAC	AGA	CAAGA
			ACCTGAATGCCCTGACAGCTCTCGCTTATACCTTCAT
hTRB	hTRB	172	ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCA	GATCC	CTCAGCC
V02	V02		GGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACA	GGTCC	ATGTACT
			CAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCAC	ACAAA	TCTGTGC
			TTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTT	GCTGG	CAGCAGT
			CTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTC	AGGA	GAAGC
			GATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTG
			AA
hTRB	hTRB	173	ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCA	CATCA	CTCTGCT
V03-1	V03-1		GGTCCCTTGGACACAGCTGTTTCCCAGACTCCAAAATACCTGGTCACA	ATTCC	GTGTATT
			CAGATGGGAAACGACAAGTCCATTAAATGTGAACAAAATCTGGGCCAT	CTGGA	TCTGTGC
			GATACTATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGATA	GCTTG	CAGCAGC
			ATGTTTAGCTACAATAATAAGGAGCTCATTATAAATGAAACAGTTCCA	GTGA	CAAGA
			AATCGCTTCTCACCTAAATCTCCAGACAAAGCTCACTTAAATCTTCA
hTRB	hTRB	174	ATGGGCTGCAGGCTCCTCTGCTATGTGGCCCTCTGCCTCCTGCAAGCA	CATCA	CTCTGCT
V03-2	V03-1		GGATCCACTGGACACAGCCGTTTCCCAGACTCCAAAATACCTGGTCAC	ATTCC	GTGTATT
			ACAGATGGGAAAAAAGGAGTCTCTTAAATGAGAACAAAATCTGGGCCA	CTGGA	TCTGTGC
			TAATGCTATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGAC	GCTTG	CAGCAGC
			AATGTTTATCTACAGTAACAAGGAGCCAATTTTAAATGAAACAGTTCC	GTGA	CAAGA
			AAATCGCTTCTCACCTGACTCTCCAGACAAAGCTCATTTAAATCTTCA
hTRB	hTRB	175	ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCA	TTCAC	AAGACTC
V04-1	V04-1		GTTCCCATAGACACTGAAGTTACCCAGACACCAAAACACCTGGTCATG	CTACA	AGCCCTG
			GGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCAC	CGCCC	TATCTCT
			AGGGCTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTC	TGCAG	GCGCCAG
			ATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGTGCCA	CCAG	CAGCCAA
			AGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACC		GA
hTRB	hTRB	176	ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCG	TTCAC	AAGACTC
V04-2	V04-2		GTCCCCATGGAAACGGGAGTTACGCAGACACCAAGACACCTGGTCATG	CTACA	GGCCCTG
			GGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGGCAT	CACCC	TATCTCT
			AACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTC	TGCAG	GTGCCAG
			ATGTTTGTCTACAACTTTAAAGAACAGACTGAAAACAACAGTGTGCCA	CCAG	CAGCCAA
			AGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATTCC		GA
hTRB	hTRB	177	ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCG	TTCAC	AAGACTC
V04-3	V04-2		GGTGAGTTGGTCCCCATGGAAACGGGAGTTACGCAGACACCAAGACAC	CTACA	GGCCCTG
			CTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACAT	CACCC	TATCTCT
			CTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCA	TGCAG	GCGCCAG
			CTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAAC	CCAG	CAGCCAA
			AGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTA		GA
			TTCC
hTRB	hTRB	178	ATGGGCTCCAGGCTGCTCTGTTGGGTGCTGCTTTGTCTCCTGGGAGCA	GAATG	GGGACTC
V05-1	V05-1		GGCCCAGTAAAGGCTGGAGTCACTCAAACTCCAAGATATCTGATCAAA	TGAGC	GGCCCTT
			ACGAGAGGACAGCAAGTGACACTGAGCTGCTCCCCTATCTCTGGGCAT	ACCTT	TATCTTT
			AGGAGTGTATCCTGGTACCAACAGACCCCAGGACAGGGCCTTCAGTTC	GGAGC	GCGCCAG
			CTCTTTGAATACTTCAGTGAGACACAGAGAAACAAAGGAAACTTCCCT	TGG	CAGCTTG
			GGTCGATTCTCAGGGCGCCAGTTCTCTAACTCTCGCTCTGAGAT		G
hTRB	hTRB	179	ATGGGCTCCGGACTCCTCTGCTGGACGCTGCTTTGTTTCCTGGGAGCA	TACTG	GGACTCA
V05-2	V05-2		GGCCCAGTGGAGGCTGGAATCACCCAAGCTCCAAGACACCTGATCAAA	AGTCA	GCCCTGT
			ACAAGAGACCAGCAAGTGACACTGAGATGCTCCCCTGCCTCTGGGCAT	AACAC	ATCTCTG
			AACTGTGTGTCCTGGTACCTACGAACTCCAAGTCAGCCCCTCTAGTTA
			TTGTTACAATATTGTAATAGGTTACAAAGAGCAAAAGGAAACTTGCCT	GGAGC	TGCCAGC
			AATTGATTCTCAGCTCACCACGTCCATAACTAT	TAGG	AACTTG
hTRB	hTRB	180	ATGGGCCCCGGGCTCCTCTGCTGGGAACTGCTTTATCTCCTGGGAGCA	GCTCT	TGGAGCT
V05-3	V05-3		GGCCCAGTGGAGGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA	GAGAT	GGGGGAC
			ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCAC	GAATG	TCGGCCC
			AGCAGTGTGTCCTGGTACCAACAGGCCCCGGGTCAGGGGCCCCAGTTT	TGAGT	TGTATCT
			ATCTTTGAATATGCTAATGAGTTAAGGAGATCAGAAGGAAACTTCCCT	GCCT	CTGTGCC
			AATCGATTCTCAGGGCGCCAGTTCCATGACTGTT		AGAAGCT
					TGG
hTRB	hTRB	181	ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCA	CTGAG	GGAGCTG
V05-4	V05-4		GGCTCAGTGGAGACTGGAGTCACCCAAAGTCCCACACACCTGATCAAA	CTGAA	GACGACT
			ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTTCTCAGTCTGGGCAC	TGTGA	CGGCCCT
			AACACTGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTT	ACGCC	GTATCTC
			ATCTTTCAGTATTATAGGGAGGAAGAGAATGGCAGAGGAAACTTCCCT	TT	TGTGCCA
			CCTAGATTCTCAGGTCTCCAGTTCCCTAATTATAGCT		GCAGCTT
					GG
hTRB	hTRB	182	ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCA	CTGAG	GTTGCTG
V05-5	V05-4		GGCCCAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA	CTGAA	GGGGACT
			ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCAC	TGTGA	CGGCCCT
			AAGAGTGTGTCCTGGTACCAACAGGTCCTGGGTCAGGGGCCCCAGTTT	ACGCC	GTATCTC
			ATCTTTCAGTATTATGAGAAAGAAGAGAGAGGAAGAGGAAACTTCCCT	TT	TGTGCCA
			GATCGATTCTCAGCTCGCCAGTTCCCTAACTATAGCT		GCAGCTT
					GG
hTRB	hTRB	183	ATGGGCCCCGGGCTCCTCTGCTGGGCACTGCTTTGTCTCCTGGGAGCA	CTGAG	GTTGCTG
V05-6	V05-4		GGCTTAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA	CTGAA	GGGGACT
			ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTAAGTCTGGGCAT	TGTGA	CGGCCCT
			GACACTGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTT	ACGCC	CTATCTC
			ATCTTTCAGTATTATGAGGAGGAAGAGAGACAGAGAGGCAACTTCCCT	TT	TGTGCCA
			GATCGATTCTCAGGTCACCAGTTCCCTAACTATAGCT		GCAGCTT
					GG
hTRB	hTRB	184	ATGGGCCCCGGGCTCCTCTGCTGGGTGCTGCTTTGTCCCCTAGGAGAA	CTGAG	GTTGCTA
V05-7	V05-4		GGCCCAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA	CTGAA	GGGGACT
			ACGAGAGGACAGCACGTGACTCTGAGATGCTCTCCTATCTCTGGGCAC	TGTGA	CGGCCCT
			ACCAGTGTGTCCTCGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTT	ACGCC	CTATCTC
			ATCTTTCAGTATTATGAGAAAGAAGAGAGAGGAAGAGGAAACTTCCCT	TT	TGTGCCA
			GATCAATTCTCAGGTCACCAGTTCCCTAACTATAGCT		GCAGCTT
					GG
hTRB	hTRB	185	ATGGGACCCAGGCTCCTCTTCTGGGCACTGCTTTGTCTCCTCGGAACA	CTGAG	GGAGCTG
V05-8	V05-4		GGCCCAGTGGAGGCTGGAGTCACACAAAGTCCCACACACCTGATCAAA	CTGAA	GAGGACT
			ACGAGAGGACAGCAAGCGACTCTGAGATGCTCTCCTATCTCTGGGCAC	TGTGA	CGGCCCT
			ACCAGTGTGTACTGGTACCAACAGGCCCTGGGTCTGGGCCTCCAGTTC	ACGCC	GTATCTC
			CTCCTTTGGTATGACGAGGGTGAAGAGAGAAACAGAGGAAACTTCCCT	TT	TGTGCCA
			CCTAGATTTTCAGGTCGCCAGTTCCCTAATTATAGCT		GCAGCTT
					GG
hTRB	hTRB	186	ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCA	GAGTT	CGGCTGC
V06-1	V06-1		AGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTG	CTCGC	TCCCTCC
			AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT	TCAGG	CAGACAT
			AACTCCATGTACTGGTATCGACAAGACCCAGGCATGGGACTGAGGCTG	CTGGA	CTGTGTA
			ATTTATTACTCAGCTTCTGAGGGTACCACTGACAAAGGAGAAGTCCCC	GT	CTTCTGT
			AATGGCTACAATGTCTCCAGATTAAACAAACGG		GCCAGCA
					GTGAAGC
hTRB	hTRB	187	ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCA	CTGGG	CCTCCCA
V06-2	V06-2		GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTG	GTTGG	AACATCT
			AAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCAT	AGTCG	GTGTACT
			GAATACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTG	GCTGC	TCTGTGC
			ATTCATTACTCAGTTGGTGAGGGTACAACTGCCAAAGGAGAGGTCCCT	TC	CAGCAGT
			GATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG		TACTC
hTRB	hTRB	188	ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCA	CTGGG	CCTCCCA
V06-3	V06-2		GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTG	GTTGG	AACATCT
			AAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCAT	AGTCG	GTGTACT
			GAATACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTG	GCTGC	TCTGTGC
			ATTCATTACTCAGTTGGTGAGGGTACAACTGCCAAAGGAGAGGTCCCT	TC	CAGCAGT
			GATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG		TACTC
hTRB	hTRB	189	ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCA	CCCCT	TACCCTC
V06-4	V06-4		GGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTG	CACGT	TCAGACA
			GCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACAT	TGGCG	TCTGTGT
			AATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTC	TCTGC	ACTTCTG
			ATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCT	TG	TGCCAGC
			GATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTC		AGTGACT
					C
hTRB	hTRB	190	ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCA	TCCCG	TGCTCCC
V06-5	V06-5		GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTG	CTCAG	TCCCAGA
			AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT	GCTGC	CATCTGT
			GAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTG	TGTCG	GTACTTC
			ATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCC	GC	TGTGCCA
			AATGGCTACAATGTCTCCAGATCAACCACAGAGGATT		GCAGTTA
					CTC
hTRB	hTRB	191	ATGAGCATCAGCCTCCTGTGCTGTGCAGCCTTTCCTCTCCTGTGGGCA	GATTT	TGGCTGC
V06-6	V06-6		GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGCATCCTG	CCCGC	TCCCTCC
			AAGATAGGACAGAGCATGACACTGCAGTGTACCCAGGATATGAACCAT	TCAGG	CAGACAT
			AACTACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAAGCTG	CTGGA	CTGTGTA
			ATTTATTATTCAGTTGGTGCTGGTATCACTGATAAAGGAGAAGTCCCG	GT	CTTCTGT
			AATGGCTACAACGTCTCCAGATCAACCACAGAG		GCCAGCA
					GTTACTC
hTRB	hTRB	192	ATGAGCCTCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCA	TCCCC	GCTCCCT
V06-7	V06-7		GGTCCAATGAATGCTGGTGTCACTCAGACCCCAAAATTCCACGTCCTG	CTCAA	CTCAGAC
			AAGACAGGACAGAGCATGACTCTGCTGTGTGCCCAGGATATGAACCAT	GCTGG	TTCTGTTT
			GAATACATGTATCGGTATCGACAAGACCCAGGCAAGGGGCTGAGGCTG	AGTCA	ACTTCTG
			ATTTACTACTCAGTTGCTGCTGCTCTCACTGACAAAGGAGAAGTTCCC	GCT	TGCCAGC
			AATGGCTACAATGTCTCCAGATCAAACACAGAGGATT		AGTTACT
					C
hTRB	hTRB	193	ATGAGCCTCGGGCTCCTGTGCTGTGCGGCCTTTTCTCTCCTGTGGGCA	TCCCA	TGCTCCC
V06-8	V06-8		GGTCCCGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCACATCCTG	CTCAG	TCCCAGA
			AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT	GCTGG	CATCTGT
			GGATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGACTG	TGTCG	GTACTTG
			ATTTACTACTCAGCTGCTGCTGGTACTACTGACAAAGAAGTCCCCAAT	GC	TGTGCCA
			GGCTACAATGTCTCTAGATTAAACACAGAGGATT		GCAGTTA
					CTC
hTRB	hTRB	194	ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGAG	GATTT	CAGCTGC
V06-9	V06-6		GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCACATCCTG	CCCGC	TCCCTCC
			AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT	TCAGG	CAGACAT
			GGATACTTGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCGC	CTGGA	CTGTATA
			ATTCATTACTCAGTTGCTGCTGGTATCACTGACAAAGGAGAAGTCCCC	GT	CTTCTGT
			GATGGCTACAATGTATCCAGATCAAACACAGAG		GCCAGCA
					GTTATTC
hTRB	hTRB	195	ATGGGCACAAGGCTCCTCTGCTGGGCAGCCATATGTCTCCTGGGGGCA	CTCTG	GCAGGGG
V07-1	V07-1		GATCACACAGGTGCTGGAGTCTCCCAGTCCCTGAGACACAAGGTAGCA	AAGTT	GACTTGG
			AAGAAGGGAAAGGATGTAGCTCTCAGATATGATCCAATTTCAGGTCAT	CCAGC	CTGTGTA
			AATGCCCTTTATTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTT	GCACA	TCTCTGT
			CCAATTTACTTCCAAGGCAAGGATGCAGCAGACAAATCGGGGCTTCCC	CA	GCCAGCA
			CGTGATCGGTTCTCTGCACAGAGGTCTGAGGGATCCATCTCCA		GCTCAGC
hTRB	hTRB	196	ATGGGCACCAGGCTCCTCTTCTGGGTGGCCTTCTGTCTCCTGGGGGCA	GATCC	GACTCGG
V07-2	V07-2		GATCACACAGGAGCTGGAGTCTCCCAGTCCCCCAGTAACAAGGTCACA	AGCGC	CCGTGTA
			GAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCAT	ACACA	TCTCTGT
			ACTGCCCTTTACTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTT	GCAGG	GCCAGCA
			TTAATTTACTTCCAAGGCAACAGTGCACCAGACAAATCAGGGCTGCCC	AG	GCTTAGC
			AGTGATCGCTTCTCTGCAGAGAGGACTGGGGGATCCGTCTCCACTCTG
			AC
hTRB	hTRB	197	ATGGGCACCAGGCTCCTCTGCTGGGCAGCCCTGTGCCTCCTGGGGGCA	ACTCT	GCGGGGG
V07-3	V07-5		GATCACACAGGTGCTGGAGTCTCCCAGACCCCCAGTAACAAGGTCACA	GAAGA	GACTCAG
			GAGAAGGGAAAATATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCAT	TCCAG	CCGTGTA
			ACTGCCCTTTACTGGTACCGACAAAGCCTGGGGCAGGGCCCAGAGTTT	CGCAC	TCTCTGT
			CTAATTTACTTCCAAGGCACGGGTGCGGCAGATGACTCAGGGCTGCCC	AGA	GCCAGCA
			AACGATCGGTTCTTTGCAGTCAGGCCTGAGGGATCCGTCTCT		GCTTAAC
hTRB	hTRB	198	ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACA	ACTCT	GCAGGGG
V07-4	V07-5		GATCACACAGGTGCTGGAGTCTCCCAGTCCCCAAGGTACAAAGTCGCA	GAAGA	GACTCAG
			AAGAGGGGACGGGATGTAGCTCTCAGGTGTGATTCAATTTCGGGTCAT	TCCAG	CTGTGTA
			GTAACCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCTCAGAGGTT	CGCAC	TCTCTGT
			CTGACTTACTCCCAGAGTGATGCTCAACGAGACAAATCAGGGCGGCCC	AGA	GCCAGCA
			AGTGGTCGGTTCTCTGCAGAGAGGCCTGAGAGATCCGTCTCC		GCTTAGC
hTRB	hTRB	199	ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACA	AGATC	GCGACTC
V07-5	V07-5		GATCACACAGGTGCTGGAGTCTCCCAGTCCCCAAGGTACGAAGTCACA	CAGCG	GGCTGTG
			CAGAGGGGACAGGATGTAGCTCCCAGGTGTGATCCAATTTCGGGTCAG	CACAG	TATCTCT
			GTAACCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCAAGAGTTT	AGCAA	GTGCCAG
			CTGACTTCCTTCCAGGATGAAACTCAACAAGATAAATCAGGGCTGCTC	GG	AAGCTTA
			AGTGATCAATTCTCCACAGAGAGGTCTGAGGATCTTTCTCCACCTGA		G
hTRB	hTRB	200	ATGGGCACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACA	CAGCG	CGGCCAT
V07-6	V07-6		GATCACACAGGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACA	CACAG	GTATCGC
			AAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCAT	AGCAG	TGTGCCA
			GTATCCCTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTT	CGGGA	GCAGCTT
			CTGACTTACTTCAATTATGAAGCCCAACAAGACAAATCAGGGCTGCCC	CT	AGC
			AATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTG
			ACGATC
hTRB	hTRB	201	ATGGGTACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACA	CAGCG	CAGCCAT
V07-7	V07-6		GATCACACAGGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACA	CACAG	GTATCGC
			AAGAGGGGACAGGATGTAACTCTCAGGTGTGATCCAATTTCGAGTCAT	AGCAG	TGTGCCA
			GCAACCCTTTATTGGTATCAACAGGCCCTGGGGCAGGGCCCAGAGTTT	CGGGA	GCAGCTT
			CTGACTTACTTCAATTATGAAGCTCAACCAGACAAATCAGGGCTGCCC	CT	AGC
			AGTGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTG
			ACGATT
hTRB	hTRB	202	ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACA	GATCC	GACTCCG
V07-8	V07-2		GATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCA	AGCGC	CCGTGTA
			AAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCAT	ACACA	TCTCTGT
			GTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTT	GCAGG	GCCAGCA
			CTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCC	AG	GCTTAGC
			AGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTG
			AA
hTRB	hTRB	203	ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCA	GAGAT	GGGACTC
V07-9	V07-9		GATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACA	CCAGC	GGCCATG
			AAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACAC	GCACA	TATCTCT
			AACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTT	GAGCA	GTGCCAG
			CTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTC	GG	CAGCTTA
			AGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTG		GC
hTRB	hTRB	204	GAGGCAGGGATCAGCCAGATACCAAGATATCACAGACACACAGGGAAA	CCCTC	GCACCAG
V08-1	V08-1		AAGATCATCCTGAAATATGCTCAGATTAGGAACCATTATTCAGTGTTC	AACCC	CCAGACC
			TGTTATCAATAAGACCAAGAATAGGGGCTGAGGCTGATCCATTATTCA	TGGAG	TCTGTAC
			GGTAGTATTGGCAGCATGACCAAAGGCGGTGCCAAGGAAGGGTACAAT	TCTAC	CTCTGTG
			GTCTCTGGAAACAAGCTCAAGCATTTT	TA	GCAGTGC
					ATC
hTRB	hTRB	205	ATGAACCCCAAACTCTTCTGTGTGACCCTTTGTCTCCTGGGAGCAGGC	TCCCC	GCACCAG
V08-2	V08-2		TCTATTGATGCTGGGATCACCCAGATGCCAAGATATCACATTGTACAG	AATCC	CCAGACC
			AAGAAAGAGATGATCCTGGAATGTGCTCAGGTTAGGAACAGTGTTCTG	TGGCA	TATCTGT
			ATATCGACAGGACCCAAGACGGGGGCTGAAGCTTATCCACTATTCAGG	TCCAC	ACCACTG
			CAGTGGTCACAGCAGGACCAAAGTTGATGTCACAGAGGGGTACTGTGT	CA	TGGCAGC
			TTCTTGAAACAAGCTTGAGCATT		ACATC
hTRB	hTRB	206	ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCA	CTAAA	GGGGGAC
V09	V09		GGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACA	CCTGA	TCAGCTT
			GCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGAC	GCTCT	TGTATTT
			CTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTC	CTGGA	CTGTGCC
			CTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTT	GCT	AGCAGCG
			GAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAA		TAG
hTRB	hTRB	207	ATGGGCACGAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCA	CCCTC	CTCCTCC
V10-1	V10-1		GGACACAGGGATGCTGAAATCACCCAGAGCCCAAGACACAAGATCACA	ACTCT	CAGACAT
			GAGACAGGAAGGCAGGTGACCTTGGCGTGTCACCAGACTTGGAACCAC	GGAGT	CTGTATA
			AACAATATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTG	CTGCT	TTTCTGC
			ATCCATTACTCATATGGTGTTCAAGACACTAACAAAGGAGAAGTCTCA	GC	GCCAGCA
			GATGGCTACAGTGTCTCTAGATCAAACACAGAGGACCTCC		GTGAGTC
hTRB	hTRB	208	ATGGGCACCAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCA	CCCTC	CCGCTCC
V10-2	V10-2		GGACACAGGGATGCTGGAATCACCCAGAGCCCAAGATACAAGATCACA	ACTCT	CAGACAT
			GAGACAGGAAGGCAGGTGACCTTGATGTGTCACCAGACTTGGAGCCAC	GGAGT	CTGTGTA
			AGCTATATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTG	CAGCT	TTTCTGC
			ATCTATTACTCAGCAGCTGCTGATATTACAGATAAAGGAGAAGTCCCC	AC	GCCAGCA
			GATGGCTATGTTGTCTCCAGATCCAAGACAGAGAATTTCC		GTGAGTC
hTRB	hTRB	209	ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACA	TCCTC	CAGCTCC
V10-3	V10-3		GGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACA	ACTCT	CAGACAT
			GAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCAC	GGAGT	CTGTGTA
			CGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTG	CCGCT	CTTCTGT
			ATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCA	AC	GCCATCA
			GATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCC		GTGAGTC
hTRB	hTRB	210	ATGAGCACCAGGCTTCTCTGCTGGATGGCCCTCTGTCTCCTGGGGGCA	CCACT	GAGCTTG
V11-1	V11-1		GAACTCTCAGAAGCTGAAGTTGCCCAGTCCCCCAGATATAAGATTACA	CTCAA	GGGACTC
			GAGAAAAGCCAGGCTGTGGCTTTTTGGTGTGATCCTATTTCTGGCCAT	GATCC	GGCCATG
			GCTACCCTTTACTGGTACCGGCAGATCCTGGGACAGGGCCCGGAGCTT	AGCCT	TATCTCT
			CTGGTTCAATTTCAGGATGAGAGTGTAGTAGATGATTCACAGTTGCCT	GCA	GTGCCAG
			AAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT		CAGCTTA
					GC
hTRB	hTRB	211	ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCA	CCACT	AAGCTTG
V11-2	V11-1		GAACTCACAGAAGCTGGAGTTGCCCAGTCTCCCAGATATAAGATTATA	CTCAA	AGGACTC
			GAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCAT	GATCC	GGCCGTG
			GCTACCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTT	AGCCT	TATCTCT
			CTGATTCAGTTTCAGAATAACGGTGTAGTGGATGATTCACAGTTGCCT	GCA	GTGCCAG
			AAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT		CAGCTTA
					GA
hTRB	hTRB	212	ATGGGTACCAGGCTCCTCTGCTGGGTGGCCTTCTGTCTCCTGGTGGAA	CCACT	GAGCTTG
V11-3	V11-1		GAACTCATAGAAGCTGGAGTGGTTCAGTCTCCCAGATATAAGATTATA	CTCAA	GGGACTC
			GAGAAAAAACAGCCTGTGGCTTTTTGGTGCAATCCTATTTCTGGCCAC	GATCC	GGCCGTG
			AATACCCTTTACTGGTACCTGCAGAACTTGGGACAGGGCCCGGAGCTT	AGCCT	TATCTCT
			CTGATTCGATATGAGAATGAGGAAGCAGTAGACGATTCACAGTTGCCT	GCA	GTGCCAG
			AAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT		CAGCTTA
					GA
hTRB	hTRB	213	ATGGGCTCTTGGACCCTCTGTGTGTCCCTTTATATCCTGGTAGCGACA	GAGGA	GGGACTT
V12-1	V12-1		CACACAGATGCTGGTGTTATCCAGTCACCCAGGCACAAAGTGACAGAG	TCCAG	GGGCCTA
			ATGGGACAATCAGTAACTCTGAGATGCGAACCAATTTCAGGCCACAAT	CCCAT	TATTTCT
			GATCTTCTCTGGTACAGACAGACCTTTGTGCAGGGACTGGAATTGCTG	GGAAC	GTGCCAG
			AATTACTTCTGCAGCTGGACCCTCGTAGATGACTCAGGAGTGTCCAAG	CCA	CAGCTTT
			GATTGATTCTCAGCACAGATGCCTGATGTATCATTCTCCACTCT		GC
hTRB	hTRB	214	ATGGACTCCTGGACCCTCTGTGTGTCCCTTTGTATCCTGGTAGCGACA	CTGAA	AGGGGGA
V12-2	V12-2		TGCACAGATGCTGGCATTATCCAGTCACCCAAGCATGAGGTGACAGAA	GATCC	CTCGGCC
			ATGGGACAAACAGTGACTCTGAGATGTGAGCCAATTTTTGGCCACAAT	AGCCT	GTGTATG
			TTCCTTTTCTGGTACAGAGATACCTTCGTGCAGGGACTGGAATTGCTG	GCAGA	TCTGTGC
			AGTTACTTCCGGAGCTGATCTATTATAGATAATGCAGGTATGCCCACA	GC	AAGTCGC
			GAGCGATTCTCAGCTGAGAGGCCTGATGGATCATTCTCTACT		TTAGC
hTRB	hTRB	215	ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCG	CAGCC	CAGCTGT
V12-3	V12-3		AAGCATACAGATGCTGGAGTTATCCAGTCACCCCGCCATGAGGTGACA	CTCAG	GTACTTC
			GAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGCCAC	AACCC	TGTGCCA
			AACTCCCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTG	AGGGA	GCAGTTT
			CTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCC	CT	AGC
			GAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTG
			AAGATC
hTRB	hTRB	216	ATGGGCTCCTGGACCCTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCA	CAGCC	CAGCTGT
V12-4	V12-3		AAGCACACAGATGCTGGAGTTATCCAGTCACCCCGGCACGAGGTGACA	CTCAG	GTACTTC
			GAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACAC	AACCC	TGTGCCA
			GACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTG	AGGGA	GCAGTTT
			CTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCC	CT	AGC
			GAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTG
			AAGATC
hTRB	hTRB	217	ATGGCCACCAGGCTCCTCTGCTGTGTGGTTCTTTGTCTCCTGGGAGAA	CAGCC	CAGCTGT
V12-5	V12-3		GAGCTTATAGATGCTAGAGTCACCCAGACACCAAGGCACAAGGTGACA	CTCAG	GTATTTT
			GAGATGGGACAAGAAGTAACAATGAGATGTCAGCCAATTTTAGGCCAC	AACCC	TGTGCTA
			AATACTGTTTTCTGGTACAGACAGACCATGATGCAAGGACTGGAGTTG	AGGGA	GTGGTTT
			CTGGCTTACTTCCGCAACCGGGCTCCTCTAGATGATTCGGGGATGCCG	CT	GGT
			AAGGATCGATTCTCAGCAGAGATGCCTGATGCAACTTTAGCCACTCTG
			AAGATC
hTRB	hTRB	218	ATGCTTAGTCCTGACCTGCCTGACTCTGCCTGGAACACCAGGCTCCTC	GAGCT	CAGCCCT
V13	V13		TGCCATGTCATGCTTTGTCTCCTGGGAGCAGTTTCAGTGGCTGCTGGA	CCTTG	GTACTTC
			GTCATCCAGTCCCCAAGACATCTGATCAAAGAAAAGAGGGAAACAGCC	GAGCT	TGTGCCA
			ACTCTGAAATGCTATCCTATCCCTAGACACGACACTGTCTACTGGTAC	GGGGG	GCAGCTT
			CAGCAGGGTCCAGGTCAGGACCCCCAGTTCCTCATTTCGTTTTATGAA	ACT	AGG
			AAGATGCAGAGCGATAAAGGAAGCATCCCTGATCGATTCTCAGCTCAA
			CAGTTCAGTGACTATCATTCTGAACTGAACAT
hTRB	hTRB	219	ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCA	GGTGC	GATTCTG
V14	V14		AAGCACATAGAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATA	AGCCT	GAGTTTA
			GAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACAT	GCAGA	TTTCTGT
			GATAATCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTT	ACTGG	GCCAGCA
			CTGTTACATTTTGTGAAAGAGTCTAAACAGGATGAGTCCGGTATGCCC	AG	GCCAAGA
			AACAATCGATTCTTAGCTGAAAGGACTGGAGGGACGTATTCTACTCTG
			AA
hTRB	hTRB	220	ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACA	GACAT	GGGACAC
V15	V15		GGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACC	CCGCT	AGCCATG
			CAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCAT	CACCA	TACCTGT
			AACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTG	GGCCT	GTGCCAC
			CTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCT	GG	CAGCAGA
			GATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTT		GA
hTRB	hTRB	221	ATGAGCCCAATATTCACCTGCATCACAATCCTTTGTCTGCTGGCTGCA	TGAGA	GAGGATT
V16	V16		GGTTCTCCTGGTGAAGAAGTCGCCCAGACTCCAAAACATCTTGTCAGA	TCCAG	CAGCAGT
			GGGGAAGGACAGAAAGCAAAATTATATTGTGCCCCAATAAAAGGACAC	GCTAC	GTATTTT
			AGTTATGTTTTTTGGTACCAACAGGTCCTGAAAAACGAGTTCAAGTTC	GAAGC	TGTGCCA
			TTGATTTCCTTCCAGAATGAAAATGTCTTTGATGAAACAGGTATGCCC	TT	GCAGCCA
			AAGGAAAGATTTTCAGCTAAGTGCCTCCCAAATTCACCCTGTAGCCT		ATC
hTRB	hTRB	222	ATGGATATCTGGCTCCTCTGCTGGGTGACCCTGTGTCTCTTGGCGGCA	GAAGA	AGGGACT
V17	V17		GGACACTCGGAGCCTGGAGTCAGCCAGACCCCCAGACACAAGGTCACC	TCCAT	CAGCCGT
			AACATGGGACAGGAGGTGATTCTGAGGTGCGATCCATCTTCTGGTCAC	CCCGC	GTATCTC
			ATGTTTGTTCACTGGTACCGACAGAATCTGAGGCAAGAAATGAAGTTG	AGAGC	TACAGTA
			CTGATTTCCTTCCAGTACCAAAACATTGCAGTTGATTCAGGGATGCCC	CG	GCGGTGG
			AAGGAACGATTCACAGCTGAAAGACCTAACGGAACGTCTTCCACGCT
hTRB	hTRB	223	ATGGACACCAGAGTACTCTGCTGTGCGGTCATCTGTCTTCTGGGGGCA	GGATC	AGATTCG
V18	V18		GGTCTCTCAAATGCCGGCGTCATGCAGAACCCAAGACACCTGGTCAGG	CAGCA	GCAGCTT
			AGGAGGGGACAGGAGGCAAGACTGAGATGCAGCCCAATGAAAGGACAC	GGTAG	ATTTCTG
			AGTCATGTTTACTGGTATCGGCAGCTCCCAGAGGAAGGTCTGAAATTC	TGCGA	TGCCAGC
			ATGGTTTATCTCCAGAAAGAAAATATCATAGATGAGTCAGGAATGCCA	GG	TCACCAC
			AAGGAACGATTTTCTGCTGAATTTCCCAAAGAGGGCCCCAGCATCCTG		C
			A
hTRB	hTRB	224	ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTTTCCTGGGAGCA	CACTG	AACCCGA
V19	V19		AACACCGTGGATGGTGGAATCACTCAGTCCCCAAAGTACCTGTTCAGA	TGACA	CAGCTTT
			AAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCAC	TCGGC	CTATCTC
			GATGCCATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTG	CCAAA	TGTGCCA
			ATCTACTACTCACAGATAGTAAATGACTTTCAGAAAGGAGATATAGCT	AG	GTAGTAT
			GAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCT		AGA
hTRB	hTRB	225	ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTA	CTGAC	CTGAAGA
V20	V20		CCTGGGAGCTTGGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACAT	AGTGA	CAGCAGC
			CCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGC	CCAGT	TTCTACA
			CGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTC	GCCCA	TCTGCAG
			CCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAG	TC	TGCTAGA
			GCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCAT		GA
			GCAAGCCTGACCTTGTCCACT
hTRB	hTRB	226	ATGTGCCTCAGACTTCTCTGCTGTGTGGCCATTTCTTTCTGGGGAGCC	GAGAT	GGGACAC
V21	V21		AGGCTCCACGGACACCAAGGTCACCCAGAGACCTAGACTTCTGGTCAA	CCAGT	AGCACTG
			AGCAAGTGAACAGAAAGCAAAGATGGATTGTGTTCCTATAAAAGCACA	CCACG	TATTTCT
			TAGTTATGTTTACTGGTATCGTAAGAAGCTGGAAGAAGAGCTCAAGTT	GAGTC	GTGCCAG
			TTTGGTTTACTTTCAGAATGAAGAACTTATTCAGAAAGCAGAAATAAT	AG	CAGCAAA
			CAATGAGCGATTTTTAGCCCAATGCTCCAAAAACTCATCCTGTACCTT		GC
			G
hTRB	hTRB	227	ATGGGGAGCTGGGTCCTCTGCTATGTGACCCTGTGTCTCCTGGGAGCA	GTGAA	AACAGCT
V22	V22		GGACCCTTGGATGCTGACATCTATCAGATGCCATTCCAGCTCACTGGG	GTTGG	TTGTACT
			GCTGGATGGGATGTGACTCTGGAGTGGAAACGGAATTTGAGACACAAT	CCCAC	TCTGTCC
			GACATGTACTGCTACTGGTACTGGCAGGACCCAAAGCAAAATCTGAGA	ACCAG	TGGGAGC
			CTGATCTATTACTCAAGGGTTGAAAAGGATATTCAGAGAGGAGATCTA	CCA	GCAC
			ACTGAAGGCTACGTGTCTGCCAAGAGGAGAAGGGGCTATTTCTTCTCA
			GG
hTRB	hTRB	228	ATGGGCACCAGGCTCCTCGGCTGTGCAGCCCTGTGTCTCCTGGCAGCA	CCTGG	CCGGGAG
V23	V23		GACTCTTTTCATGCCAAAGTCACACAGACTCCAGGACATTTGGTCAAA	CAATC	ACACGGC
			GGAAAAGGACAGAAAACAAAGATGGATTGTACCCCCGAAAAAGGACAT	CTGTC	ACTGTAT
			ACTTTTGTTTATTGGTATCAACAGAATCAGAATAAAGAGTTTATGCTT	CTCAG	CTCTGCG
			TTGATTTCCTTTCAGAATGAACAAGTTCTTCAAGAAACGGAGATGCAC	AA	CCAGCAG
			AAGAAGCGATTCTCATCTCAATGCCCCAAGAACGCACCCTGCAG		TCAATC
hTRB	hTRB	229	ATGGCCTCCCTGCTCTTCTTCTGTGGGGCCTTTTATCTCCTGGGAACA	GAGTC	CAGCTCT
V24	V24		GGGTCCATGGATGCTGATGTTACCCAGACCCCAAGGAATAGGATCACA	TGCCA	TTACTTC
			AAGACAGGAAAGAGGATTATGCTGGAATGTTCTCAGACTAAGGGTCAT	TCCCC	TGTGCCA
			GATAGAATGTACTGGTATCGACAAGACCCAGGACTGGGCCTACGGTTG	AACCA	CCAGTGA
			ATCTATTACTCCTTTGATGTCAAAGATATAAACAAAGGAGAGATCTCT	GA	TTTG
			GATGGATACAGTGTCTCTCGACAGGCACAGGCTAAATTCTCCCTGTCC
			CTA
hTRB	hTRB	230	ATGACTATCAGGCTCCTCTGCTACATGGGCTTTTATTTTCTGGGGGCA	GGAGT	TACCTCT
V25	V25		GGCCTCATGGAAGCTGACATCTACCAGACCCCAAGATACCTTGTTATA	CTGCC	CAGTACC
			GGGACAGGAAAGAAGATCACTCTGGAATGTTCTCAAACCATGGGCCAT	AGGCC	TCTGTGC
			GACAAAATGTACTGGTATCAACAAGATCCAGGAATGGAACTACACCTC	CTCAC	CAGCAGT
			ATCCACTATTCCTATGGAGTTAATTCCACAGAGAAGGGAGATCTTTCC	A	GAATA
			TCTGAGTCAACAGTCTCCAGAATAAGGACGGAGCATTTTCCCCTGACC
			CT
hTRB	hTRB	231	ATGAGCAACAGGCTTCTCTGCTGTGTGATCATTTGTCTCCTAAGAGCA	GAAGT	ACATCTG
V26	V26		GGCCTCAAGGATGCTGTAGTTACACAATTCCCAAGACACAGAATCATT	CTGCC	TGTATCT
			GGGACAGGAAAGGAATTCATTCTACAGTGTTCCCAGAATATGAATCAT	AGCAC	CTATGCC
			GTTACAATGTACTGGTATCGACAGGACCCAGGACTTGGACTGAAGCTG	CAACC	AGCAGTT
			GTCTATTATTCACCTGGCACTGGGAGCACTGAAAAAGGAGATATCTCT	AG	CATC
			GAGGGGTATCATGTTTCTTGAAATACTATAGCATCTTTTCCCCTGACC
			CT
hTRB	hTRB	232	ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCA	GGAGT	ACCTCTC
V27	V27		GGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACA	CGCCC	TGTACTT
			GTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCAT	AGCCC	CTGTGCC
			GAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAG	CAACC	AGCAGTT
			ATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCT	AG	TATC
			GAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATC
			CT
hTRB	hTRB	233	ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTA	GGAGT	ACATCTA
V28	V28		GGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAA	CCGCC	TGTACCT
			AGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCAT	AGCAC	CTGTGCC
			GAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTG	CAACC	AGCAGTT
			ATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCT	AG	TATG
			GAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATT
			CT
hTRB	hTRB	234	ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGT	GTGAG	CAGCAGC
V29	V29		GCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACC	CAACA	ATATATC
			TCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTC	TGAGC	TCTGCAG
			TGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCA	CCTGA	CGTTGAA
			AATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAG	AGA	GA
			TTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACT
hTRB	hTRB	235	ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTC	GAGTT	GTGACTC
V30	V30		AGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTG	CTAAG	TGGCTTC
			GGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCC	AAGCT	TATCTCT
			AACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTC	CCTTC	GTGCCTG
			TTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAAT	TCA	GAGTGT
			CTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCT