WO2013109935A1 - Method for analysis of immune variable sequences - Google Patents

Abstract

The immune system responds to disease by inducing cellular responses. Methods for sequencing and analysis of immune repertoires can be used to develop noninvasive diagnostics, high-value diagnostics that inform treatment regimens, and novel therapeutic agents. However, immune repertoires have up to 106 diversity, so computer analysis can be slow and error prone. In this invention, we identify unique immune repertoire reference sequences "words" that unambiguously identify genes and/or subgroups of genes, and then use these words to analyze multiplexed immune repertoire data.

Description

METHOD FOR ANALYSIS OF IMMUNE VARIABLE SEQUENCES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Appn. No. 61/588,878 filed January 20, 2012, which is hereby incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

[0002] This invention relates generally to immune repertoire specific reference sequences ("words") that unambiguously identify genes and/or subgroups of genes, and the use of these words to analyze multiplexed immune repertoire data.

2. BACKGROUND OF THE INVENTION

2.1. Introduction

[0003] Immune systems are comprised of a huge diversity of immune cells, such as T cells and B cells. Immune cell repertoires are comprised of millions of clones, which produce proteins that enable each cell to specifically recognize a single antigen. When the cells recognize that antigen, they produce an immune response. Genetic analysis of millions of immune cells is useful in medicine and research, in part because components of an individual's immune system are indicative of health. Disregulation of the immune system is responsible for a variety of disorders including autoimmune diseases such as Crohn's disease, juvenile diabetes (Type 1 diabetes, TID), multiple sclerosis, rheumatoid arthritis, and systemic lupus erythromatosis (SLE). Immune monitoring is useful to better understand cancer, immunotherapy, and immune-competence. In addition, detailed analysis of the immune system can determine appropriate donors for organ transplants and monitor for signs of graft versus host disease (GVHD).

[0004] Antibodies are produced by recombined genomic immunoglobulin (Ig) sequences in B lineage cells. Immunoglobulin light chains are derived from either /cor λ genes. The λ genes are comprised of four constant (C) genes and approximately thirty variable (V) genes. In contrast, the κ genes are comprised of one C gene and 250 V genes. The heavy chain gene family is comprised of several hundred V genes, fifteen D genes, and four joining (J) genes. Somatic recombination during B cell differentiation randomly chooses one V-D-J combination in the heavy chain and one V-J combination in either /cor λ light chain. Because there are so many genes, millions of unique combinations are possible. The V genes also undergo somatic hypermutation after recombination, generating further diversity. Despite this underlying complexity, it is possible to use dozens of primers targeting conserved sequences to sequence the full heavy and light chain complement in several multiplexed reactions (van Dongen et al, 2003 Leukemia 17: 2257-2317).

[0005] T cells use T cell receptors (TCR) to recognize antigens and control immune responses. The T cell receptor is composed of two subunits: a and β or γ and δ. Much of the peptide variability of the TCR is encoded in complementary determining region 3β (CDR3P), which is formed by recombination between noncontiguous variable (V), diversity (D), and joining (J) genes in the b chain loci (Wang et al, 2010 PNAS 107: 1518-23). A published set of forty-five forward primers and thirteen reverse primers amplify the ~200bp recombined genomic CDR3P region for multiplex amplification of the full CDR3P complement of a sample of human peripheral blood mononuclear cells (Robins et al, 2009 Blood 114:4099- 4107; Robins et al., 2010 Science Translational Med 2:47ra64). The CDR3 region begins with the second conserved cysteine in the 3' region of the νβ gene and ends with the conserved phenylalanine encoded by the 5' region of the Ιβ gene (Monod et al., 2004 Bioinformatics 20:i379-i385). Thus, amplified sequences can be informatically translated to locate the conserved cysteine, obtain the intervening peptide sequence, and tabulate counts of each unique clone in the sample.

[0006] Several patent applications have published disclosing molecular methods for multiplexed immune repertoire analysis by PCR and deep sequencing. Han (WO 2009/137255) describes a protocol and primer system for amplification of immune repertoires. Lim et al. (WO 2005/059176) also describes a very similar multiplexed method. Fahem & Willis (WO 2010/053587) describes a molecular system and method for multiplexed molecular analysis of immune repertoires that is similar to Han and Lim. However, none of these disclosures describe a computational method for the rapid and accurate processing of immune repertoire data.

[0007] The data sets produced by multiplexed PCR or next generation sequencing are intrinsically complex and require advanced informatic processing. The current invention discloses a robust, fast, and accurate computational method for processing immune repertoire data.

3. SUMMARY OF THE INVENTION

[0008] In particular non- limiting embodiments, the present invention provides a method for immune repertoire sequence identification which comprises: comparing an unknown sequence to a plurality of immune repertoire specific reference sequences; if a portion of the unknown sequence matches an immune repertoire reference sequence, measuring a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence; and using the measured distance and the immune repertoire reference sequence to identify the unknown sequence.

[0009] In addition, the invention provides a computer-implemented method for immune repertoire sequence identification which comprises: comparing an unknown sequence to a plurality of immune repertoire specific reference sequences; if a portion of the unknown sequence matches an immune repertoire reference sequence, measuring a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence; and using the measured distance and the immune repertoire reference sequence to identify the unknown sequence.

[0010] Furthermore, the invention provides a system for immune repertoire sequence identification which comprises: an alignment module wherein an unknown sequence is aligned with a plurality of immune repertoire specific reference sequences; and a measurement module that measures and compares a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown.

4. BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1. General schematic / flow chart with boxes/steps of the method of using the immune repertoire specific reference sequences.

[0012] Figure 2. Example of immune repertoire specific reference sequences unique to TCR V and J genes used to identify V (TRBV19) and J (TRBJ1-2) genes in an unknown sequence. The V tag is used to identify the start of the CDR3P peptide. (SEQ ID NOS:l-3)

[0013] Figure 3. Another example of immune repertoire specific reference sequences unique to TCR V and J genes used to identify V (TRBV6-6) and J (TRBJ2-7) genes in an unknown sequence. Here, two immune repertoire specific reference sequences are used to identify the TRBV6-6 genes. (SEQ ID NOS:4-6)

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

[0014] Terms used in the claims and specification are defined as set forth below unless otherwise specified. [0015] The term "B cell" refers to a type of lymphocyte that plays a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells). The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells (APCs) and eventually develop into memory B cells after activation by antigen interaction. B cells are an essential component of the adaptive immune system.

[0016] The term "bulk sequencing" or "next generation sequencing" or "massively parallel sequencing" refers to any high throughput sequencing technology that parallelizes the DNA sequencing process. For example, bulk sequencing methods are typically capable of producing more than one million polynucleic acid amplicons in a single assay. The terms "bulk sequencing," "massively parallel sequencing," and "next generation sequencing" refer only to general methods, not necessarily to the acquisition of greater than 1 million sequence sequences in a single run. Any bulk sequencing method can be implemented in the invention, such as reversible terminator chemistry (e.g., Illumina), pyrosequencing using polony emulsion droplets (e.g., Roche), ion semiconductor sequencing (IonTorrent), single molecule sequencing (e.g., Pacific Biosciences), massively parallel signature sequencing, etc.

[0017] The term "cell" refers to a functional basic unit of living organisms. A cell includes any kind of cell (prokaryotic or eukaryotic) from a living organism. Examples include, but are not limited to, mammalian mononuclear blood cells, yeast cells, or bacterial cells.

[0018] The term "gene" refers to a nucleic acid sequence that can be potentially transcribed and/or translated which may include the regulatory elements in 5' and 3', and the introns, if present. Examples of genes are TRBV10-6, TRBJ2-7. See "gene" at www.imgt.org.

[0019] The term "group" a set of genes which share the same gene type and participate potentially to the synthesis of a polypeptide of the same immunologic chain type. By extension, a group includes the related pseudogenes and orphans. A group is independent from the species. Groups are defined for the immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) molecules, e.g. , TRBJ, TRBV and TRBD are part of the same group. See "group" at www.imgt.org.

[0020] The term "ligase chain reaction" or LCR refers to a type of DNA amplification where two DNA probes are ligated by a DNA ligase, and a DNA polymerase is used to amplify the resulting ligation product. Traditional PCR methods are used to amplify the ligated DNA sequence.

[0021] The term "mammal" as used herein includes both humans and non-humans and include, but is not limited to, humans, non-human primates, canines, felines, murines, bo vines, equines, and porcines.

[0022] The term "polymerase chain reaction" or PCR refers to a molecular biology technique for amplifying a DNA sequence from a single copy to several orders of magnitude (thousands to millions of copies). PCR relies on thermal cycling, which requires cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments, or oligonucleotides) containing sequences complementary to the target region of the DNA sequence and a DNA polymerase are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. A heat- stable DNA polymerase, such as Taq polymerase, is used. The thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

[0023] The term "reverse transcriptase polymerase chain reaction" or RT-PCR refers to a type of PCR reaction used to generate multiple copies of a DNA sequence. In RT-PCR, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional PCR techniques.

[0024] The term "subgroup" refers to a set of IG or TR genes (C-gene, V-gene, D-gene or J-gene) which belong to the same group, in a given species, and which share at least 75% identity at the nucleotide level (in the germline configuration for V, D, and J), e.g. , TRBV6-1 and TRBV6-2 are genes in the TRBV6 subgroup. See "subgroup" in www.imgt.org.

[0025] The term "T cell" refers to a type of cell that plays a central role in cell-mediated immune response. T cells belong to a group of white blood cells known as lymphocytes and can be distinguished from other lymphocytes, such as B cells and natural killer T (NKT) cells by the presence of a T cell receptor (TCR) on the cell surface. T cells responses are antigen specific and are activated by foreign antigens. T cells are activated to proliferate and differentiate into effector cells when the foreign antigen is displayed on the surface of the antigen-presenting cells in peripheral lymphoid organs. T cells recognize fragments of protein antigens that have been partly degraded inside the antigen-presenting cell. There are two main classes of T cells - cytotoxic T cells and helper T cells. Effector cytotoxic T cells directly kill cells that are infected with a virus or some other intracellular pathogen. Effector helper T cells help to stimulate the responses of other cells, mainly macrophages, B cells and cytotoxic T cells.

[0026] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

5.2. GENERAL METHODS

[0027] In one embodiment, the current invention is a method for analysis of repertoires of immune variable sequences, in groups such as IG and TR. This invention has broad applicability in many areas of biological analysis. The useful applications can include cancer diagnostics, immunology, or infectious disease diagnostics.

[0028] In particular non- limiting embodiments, the present invention provides a method for immune repertoire sequence identification which comprises: comparing an unknown sequence to a plurality of immune repertoire specific reference sequences; if a portion of the unknown sequence matches an immune repertoire reference sequence, measuring a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence; and using the measured distance and the immune repertoire reference sequence to identify the unknown sequence.

[0029] In one embodiment, multiplexed polymerase chain reaction is used to amplify the unknown sequence. In another embodiment, massively parallel sequencing is used to sequence the unknown sequence. In yet another embodiment, the immune repertoire specific reference sequences are immunoglobulin IgH, immunoglobulin IgL, T cell receptor (TCR ) or T cell receptor (TCRoc) reference sequences.

[0030] In one embodiment, the immune repertoire specific reference sequences are joining (J) gene or variable (V) gene reference sequences. In some embodiments, the conserved reference codon is a second conserved cysteine, a phenylalanine or a tryptophan codon. In other embodiments, the immune repertoire specific reference sequence or the conserved reference codon are selected from the sequences in Table 1 or the distance to the conserved reference codon is a distance selected from the distances to the reference codons in Table 1.

[0031] In addition, the invention provides a computer-implemented method for immune repertoire sequence identification which comprises: comparing an unknown sequence to a plurality of immune repertoire specific reference sequences; if a portion of the unknown sequence matches an immune repertoire reference sequence, measuring a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence; and using the measured distance and the immune repertoire reference sequence to identify the unknown sequence.

[0032] Furthermore, the invention provides a system for immune repertoire sequence identification which comprises: an alignment module wherein an unknown sequence is aligned with a plurality of immune repertoire specific reference sequences; and a measurement module that measures and compares a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown.

[0033]

5.3. USE OF THE METHODS

[0034] Methods of the invention are applied to post-transplant immune monitoring whether autologous, allogeneic, syngeneic, or xenographic. After an allogeneic transplant (i.e. , kidney, liver, or stem cells), a host's T cells response to transplants are assessed to monitor the health of the host and the graft. Molecular monitoring of blood or urine is helpful to detect acute or chronic rejection before a biopsy would typically be indicated. For example, detection of alloantibodies to human leukocyte antigen (HLA) has been associated with chronic allograft rejection (Terasaki and Ozawa, 2004 American Journal of Transplantation 4:438-43). Other molecular markers include b2-microglobulin, neopterin, and proinflammatory cytokines in urine and blood (Sabek et ah , 2002 Transplantation 74:701-7; Tatapudi et ah , 2004 Kidney International 65:2390; Matz et ah , 2006 Kidney International 69: 1683; Bestard et ah , 2010 Current Opinion in Organ Transplantation 15:467-473). However, none of these methods has become widely adopted in clinical practice, perhaps due to low specificity and sensitivity. Prior work has shown that regulatory T cells (Treg) induce graft tolerance by down-regulating helper T cells (Th) (Graca et ah , 2002 Journal of Experimental Medicine 195: 1641). Additionally, transplanting hematopoietic stem cells from HLA-mismatched donors into the recipient has resulted in long-term nonimmunosuppressive renal transplant tolerance up to 5 years after transplant (Kawai et al , 2008 NEJM 358:353-61).

5.4. T Cell Analysis and Latent Tuberculosis Diagnosis

[0035] Latent tuberculosis (TB) is a major global epidemic, affecting as many as 2 billion people worldwide. There is currently no reliable test for clinical diagnosis of latent TB. This technology gap has severe clinical consequences, since reactivated TB is the only reliable hallmark of latent TB. Furthermore, clinical trials for vaccines and therapies lack biomarkers for latent TB, and therefore must follow cohorts over many years to prove efficacy.

[0036] The major current vaccine for tuberculosis, bacillus Calmette-Guerin (BCG), is an unreliable prophylactic. In a meta-analysis of dozens of epidemiological studies, the overall effect of BCG was 50% against TB infections, 78% against pulmonary TB, 64% against TB meningitis, and 71% against death due to TB infection (Colditz et al , 1994 JAMA 271 :698- 702). Additionally, the rapid rise in multidrug resistant TB has increased the need for new vaccine and immunotherapy approaches. Up to 90% of infected, immunocompetent individuals never progress to disease, resulting in the huge global latent TB reservoir (Kaufmann, 2005 Trends in Immunology 26:660-67).

[0037] Since tuberculosis is a facultative intracellular pathogen, immunity is almost entirely mediated through T cells. Interferon-g expressing T helper 1 (Thl) cells elicit primary TB response, with some involvement by T helper 2 cells (Th2). After primary response, the bacteria become latent, controlled by regulatory T cell (Treg) and memory T cells (Tmem). Recently, eleven new vaccine candidates have entered clinical trials (Kaufmann, 2005 Trends in Immunology 26:660-67). These vaccines are all "post-exposure" vaccines, i.e. , they target T cell responses to latent TB and are intended to prevent disease reactivation. Because of the partial failure of BCG to induce full immunity, rational design and validation of future TB vaccines should include systematic analysis of the specific immune response to both TB and the new vaccines.

[0038] For decades, the standard of care for diagnosis of latent tuberculosis has been the tuberculin skin test (TST) (Pai et al , 2004 Lancet Infectious Disease 4:761-76). More recently, two commercial in vitro interferon-g assays have been developed: the QuantiFERON-TB assay and the T SPOT-TB assay. These assays measure cell-mediated immunity by quantifying interferon-g released from T cells when challenged with a cocktail of tuberculosis antigens. Unfortunately, neither the TST nor the newer interferon-g tests is effective at distinguishing latent TB from cleared TB (Diel et al. , 2007 American Journal of Respir Crit Care Med 177: 1164-70). This is a significant problem because patients without clinical evidence of latent TB {i.e. , visualization of granulomas) but with positive TST or interferon-g test typically receive 6-9 months of isoniazide therapy, even though this empiric intervention is unnecessary in patients who have cleared primary infection and can cause serious complications such as liver failure.

[0039] Prior work has demonstrated that T cell responses are used to distinguish latent from active TB (Schuck et al , 2009 PLoS One 4:e5590). The premise of this prior work is that immune cells directed against TB antigens will be expanded in the memory T cell population if the TB is latent, but expanded in a helper T cell fraction if the TB is active. Functional T cell sequencing is used to distinguish latent TB from cleared TB.

5.5. T Cell Analysis and Diagnosing or Monitoring Disease

[0040] Similarly, functional T cell monitoring is used for diagnosis and monitoring of nearly any human disease. These diseases, include but are not limited to, systemic lupus erythmatosis (SLE), allergy, autoimmune disease, heart transplants, liver transplants, bone marrow transplants, lung transplants, solid tumors, liquid tumors, myelodysplastic syndrome (MDS), chronic infection, acute infection, hepatitis, human papilloma virus (HPV), herpes simplex virus, cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Such monitoring includes individual diagnosis and monitoring or population monitoring for epidemiological studies.

[0041] T cell monitoring is used for research purposes using any non-human model system, such as zebrafish, mouse, rat, or rabbit. T cell monitoring also is used for research purposes using any human model system, such as primary T cell lines or immortal T cell lines.

5.6. B Cell Analysis and Drug Discovery

[0042] Antibody therapeutics are increasingly used by pharmaceutical companies to treat intractable diseases such as cancer (Carter 2006 Nature Reviews Immunology 6:343-357). However, the process of antibody drug discovery is expensive and tedious, requiring the identification of an antigen, and then the isolation and production of monoclonal antibodies with activity against the antigen. Individuals that have been exposed to disease produce antibodies against antigens associated with that disease. Thus, it is possible mine patient immune repertoires for specific antibodies that could be used for pharmaceutical development.

5.7. B Cell Analysis and Monitoring Immunity

[0043] Humoral memory B cells (Bmem) help mammalian immune systems retain certain kinds of immunity. After exposure to an antigen and expansion of antibody-producing cells, Bmem cells survive for many years and contribute to the secondary immune response upon re-introduction of an antigen. Such immunity is typically measured in a cellular or antibody- based in vitro assay. In some cases, it is beneficial to detect immunity by amplifying, linking, and detecting IgH and light chain immunoglobulin variable regions in single B cells. Such a method is more specific and sensitive than current methods. Massively parallel B cell repertoire sequencing is used to screen for Bmem cells that contain a certain heavy and light chain pairing which is indicative of immunity.

5.8. B Cell Analysis and Diagnosing and Monitoring Disease

[0044] B cell monitoring is used for diagnosis and monitoring of nearly any human disease. These diseases include, but are not limited to, systemic lupus erythmatosis (SLE), allergy, autoimmune disease, heart transplants, liver transplants, bone marrow transplants, lung transplants, solid tumors, liquid tumors, myelodysplastic syndrome (MDS), chronic infection, acute infection, hepatitis, human papilloma virus (HPV), herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Such monitoring could include individual diagnosis and monitoring or population monitoring for epidemiological studies.

[0045] B cell monitoring is also used for research purposes using any non-human model system, such as zebrafish, mouse, rat, or rabbit. B cell monitoring is used for research purposes using any human model system, such as primary B cell lines or immortal B cell lines.

[0046] The following Examples further illustrate the invention and are not intended to limit the scope of the invention.

6. EXAMPLES [0047] Here is a method for TCR repertoire identification. Because the TCR repertoire contains as many as 5xl0⁶ clonotypes, and CDR3 regions often differ by only a few nucleotides, a sophisticated custom analysis platform is necessary just to identify the clones in the library. Turnkey fast-alignment methods such as BLAST (Altschul et al., 1990 J Mol Biol 215:403-410), BLAT (Kent 2002 Genome Research 12:656-64), and SOAP (Li et al, 2008, Bioinformatics 24:713-4) are inadequate for the task at hand, because they result in many spurious matches. Moreover, highly accurate turnkey methods such as the Smith- Waterman Algorithm (Smith and Waterman, 1981, Journal of Molecular Biology 147: 195- 197) are cumbersomely slow for this kind of analysis. Corbett et al. report a Germline Query (GQ) a program that uses BLAST and sequential queries for V genes, J genes, and D genes (Corbett et al., 1997, Journal of Molecular Biology 270: 587-597). The National Center for Biotechnology Information (NCBI) has a BLAST tool for immune sequences, IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/). Finally, all of these methods would require a huge reference library (10¹⁵ diversity) of all possible CDR3 nucleotide sequences, which is a computational burden.

[0048] Specifically, common sequence aligners such as BLAST/BLAT, bwa (Burrows- Wheeler transform, Li and Durbin 2009 Bioinformatics, 25, 1754-1760), and SOAP allow sequences to contain errors while aligning them to a reference. While this is of advantage when aligning a large number of reads (for example in evolutionary biology), for immune repertoire analysis, this feature is undesirable.

[0049] The V and J gene sequences are very similar to each other within their group, often showing as little as one base difference in the CDR3 region. For example, TRBV12-3 and TRBV12-4 are identical over 97.7% of 347 bases; TRBV6-2 and TRBV6-3 are 99.7% identical over 344 bases. For repertoire analysis, one needs exact identification of the genes or subgroups of genes and cannot tolerate errors in the alignment.

[0050] To address these problems, this invention describes a method significantly faster than any current methods and which has the same accuracy as standard alignment methods. The method starts with a table of nucleotide "words" often 4-23 base word or word pairs that uniquely identify the V and J genes of mouse or human within the amplified region. Next, the validity of each V gene match is tested by identifying the distance to and the sequence of a conserved codon, e.g., the second conserved cysteine in the case of TCR . The match is accepted as correct only if both distance and cysteine sequence confirm the match. Using data from our TCR repertoire sequencing experiments, typically -99.98% of V-Ιβ combinations are identified unambiguously. The remaining reads are discarded.

[0051] In non-limiting embodiments there are two optional further quality control steps: (i) requiring that the CDR3 region must not contain any sequencing errors in the form of uncalled bases; or (ii) requiring that the CDR3 region is in frame as defined by the second conserved cysteine. In one embodiment, only if all quality tests are passed, the method identifies the protein coding sequence of the CDR3 region within the known reading frame for that particular gene. Some input sequences in the method may contain errors. To minimize our susceptibility to errors, the uniquely identifying words are as short as possible, therefore reducing the probability of identifying a gene incorrectly. This method ensures speed, accuracy and lowest error rates. The method may be used readily for other variable gene families, such as TCRa, HLA, or IgH.

[0052] The immune repertoire specific sequences, or "words", are unique only in the area of and around the CDR3 region that is amplified, but not over the entire V or J genes (which are several hundred bases long). In one embodiment, the sequences are amplified with a method similar to Robins et al. Blood 114:4099-4107: "The Vbeta forward primer is anchored at position -43 in the Vbeta segment, relative to the recombination signal sequence. [...] The Jbeta reverse primers were designed to be anchored at their 3' ends on a consensus splice site motif."

[0053] The optimized words may be longer than 4-8 bases. For human, the J-words are 4- 6 bases long; the V-words come in singles and pairs; the shortest single is 5 bases long, the longest single is 15 bases long; the shortest pair is 19 bases and the longest pair is 23 bases long. Table 1 below provides a complete set of immune specific reference sequences, "words" for human TCR and exemplary words for human TCRa, human IgH.

[0054] For mouse the V-words range from 6-13 bases with only one pair of 10 bases, the J-words range from 4-6 bases.

[0055] Words were optimized to minimize misidentification across genes, minimize their length (hence pairs and not one long one), and maximize "Hamming distance" to other words (Hamming, 1950, Bell System Tech. J. 29: 147-160). The human V-words have to be longer since there are large families (e.g. 6, 12) of V genes with very similar sequences. There are 3 pairs of V-genes that cannot be uniquely identified using this method, because their V and J genes are identical within the CDR3 region: TRBV12-3 and TRBV12-4, TRBV6-2 and TRBV6-3, and TRBV6-5 and TRBV6-6. In agreement with standard practice the method attributes the occurrence of either sequence to only TRBV12-4, TRBV6-3, and TRBV6-6.

[0056] The computational complexity for Smith-Waterman (SW) alignment of two sequences of length m and n is O(nm). SW aligns 76 base reads against 13 J-genes of median length of 21 at 20,748 time units per read. SW aligns 76 base reads against 50 V-genes of median length of 38 at 144,400 time units per read. The method described herein aligns 6 bases against 13 J-words of length 4 at 312 time units per read. The method described herein aligns 43 bases against 50 V-words of median length 9 at 19,350 time units per read.

[0057] Therefore, the J gene alignment is 66.5x faster than SW and the V gene 7.5x faster than SW.

[0058] The total processing cost is 165,148 time units per read for SW and 19,662 time units per read for the method described herein, making it 8.4x faster than SW. Preferably, one has to have the shortest possible words, while maintaining the greatest possible difference between them.

[0059] TABLE 1 lists exemplary sets of (i) gene names, (ii) the immune repertoire specific sequences, (iii) the nucleotide sequence for the conserved codons, and (iv) the positive (+) or negative (-) distances to the conserved codon. For some genes two immune repertoire specific sequences are preferred for use in the methods described herein which are separated by a space e.g., TRVB4-2. One of ordinary skill in the art will recognize that one readily could use the complementary sequence in the methods disclosed herein.

TABLE 1

[0060] The deposited sequence listing also provides complete sets of sequences for human and murine TCRp, TCRa, and IgH J and V genes (SEQ ID NOS: 41-595). See Table 2 below for the mapping of the SEQ ID NO to the sequence names. One of ordinary skill could readily obtain such sequences from databases such as RefSeq (http://www.ncbi.nlm.nih.gov/gene/), the international ImMunoGeneTics information system® (http://www.imgt.org/), EMBL Nucleotide Sequence Database VBASE2 (http://www.vbase2.org/), or MRC Centre for Protein Engineering V BASE (http://vbase.mrc-cpe.cam.ac.uk/).

6.1. COMPUTER IMPLEMENTED METHODS

[0061] The computer-implemented method or system may be configured in either hardware, software, or both based on the types of applications needed and the hardware available. Hardware examples of implementation include hardware implemented ASIC ("Application Specific Integrated Circuit"), SOC ("System on a Chip"), RISC ("Reduced Instruction Set Computing") processor, general processor, DSP ("Digital Signal Processor"), etc.

[0062] The various implementations of the subject matter disclosed herein may be implemented in hardware, software, or both. In the present context, software comprises an ordered listing of executable instructions for implementing logical functions, and may selectively be embodied in any computer-readable medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, processor-containing system, or other system that may selectively fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions. In the context of this document, a "computer-readable medium" is any means that may contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. The computer-readable medium may selectively be, for example but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific (yet a non-exhaustive list of) examples of the computer-readable medium would include the following: an electrical connection (electronic) having one or more wires, a portable computer diskette (magnetic), a RAM (electronic), a read-only memory "ROM" (electronic), an erasable programmable read-only memory (EPROM or Flash memory) (electronic), an optical fiber (optical), and a portable compact disc read-only memory "CDROM" (optical).

[0063] While in the foregoing detailed description this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

[0064] It also is to be understood that, while the invention has been described in conjunction with the detailed description, thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications of the invention are within the scope of the claims set forth below. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

TABLE 2

77 >IgHV3-66

78 >IgHV3-72

79 >IgHV3-73

80 >IgHV3-74

81 >IgHV4-b

82 >IgHV4-4

83 >IgHV4-28

84 >IgHV4-30-2

85 >IgHV4-30-4

86 >IgHV4-31

87 >IgHV4-34

88 >IgHV4-39

89 >IgHV4-59

90 >IgHV4-61

91 >IgHV5-a

92 >IgHV5-51

93 >IgHV6-l

94 >IgHV7-4-l

>gi 1224589805 : 23012122-23012183 Homo sapiens chromosome 14, GRCh37.p5

95 Primary Assembly, TRAJ3

>gi 1224589805 : 23011142-23011204 Homo sapiens chromosome 14, GRCh37.p5

96 Primary Assembly, TRAJ4

>gi 1224589805 : 23009190-23009249 Homo sapiens chromosome 14, GRCh37.p5

97 Primary Assembly, TRAJ5

>gi 1224589805 : 23008016-23008077 Homo sapiens chromosome 14, GRCh37.p5

98 Primary Assembly, TRAJ6

>gi 1224589805 : 23006567-23006625 Homo sapiens chromosome 14, GRCh37.p5

99 Primary Assembly, TRAJ7

>gi 1224589805 : 23005092-23005151 Homo sapiens chromosome 14, GRCh37.p5

100 Primary Assembly, TRAJ8

>gi 1224589805 : 23004502-23004562 Homo sapiens chromosome 14, GRCh37.p5

101 Primary Assembly, TRAJ9

>gi 1224589805 : 23002445-23002508 Homo sapiens chromosome 14, GRCh37.p5

102 Primary Assembly, TRAJ10

>gi 1224589805 : 23001452-23001511 Homo sapiens chromosome 14, GRCh37.p5

103 Primary Assembly, TRAJll

>gi 1224589805 : 23000889-23000948 Homo sapiens chromosome 14, GRCh37.p5

104 Primary Assembly, TRAJ12

>gi 1224589805 : 23000026-23000088 Homo sapiens chromosome 14, GRCh37.p5

105 Primary Assembly, TRAJ13

>gi 1224589805 : 22999278-22999329 Homo sapiens chromosome 14, GRCh37.p5

106 Primary Assembly, TRAJ14

>gi 1224589805 : 22998580-22998641 Homo sapiens chromosome 14, GRCh37.p5

107 Primary Assembly, TRAJ15

>gi 1224589805 : 22997487-22997546 Homo sapiens chromosome 14, GRCh37.p5

108 Primary Assembly, TRAJ16

>gi 1224589805 : 22995812-22995874 Homo sapiens chromosome 14, GRCh37.p5

109 Primary Assembly, TRAJ17

>gi 1224589805 : 22994620-22994685 Homo sapiens chromosome 14, GRCh37.p5

110 Primary Assembly, TRAJ18

>gi 1224589805 : 22993296-22993352 Homo sapiens chromosome 14, GRCh37.p5

111 Primary Assembly, TRAJ20

112 >gi 1224589805 : 22992573-22992627 Homo sapiens chromosome 14, GRCh37.p5 Primary Assembly, TRAJ21

>gi 1224589805:22991016-22991078 Homo sapiens chromosome 14, GRCh37 p5

113 Primary Assembly, TRAJ22

>gi 1224589805:22989394-22989456 Homo sapiens chromosome 14, GRCh37 p5

114 Primary Assembly, TRAJ23

>gi 1224589805:22988947-22989009 Homo sapiens chromosome 14, GRCh37 p5

115 Primary Assembly, TRAJ24

>gi 1224589805:22987424-22987483 Homo sapiens chromosome 14, GRCh37 p5

116 Primary Assembly, TRAJ26

>gi 1224589805:22985251-22985309 Homo sapiens chromosome 14, GRCh37 p5

117 Primary Assembly, TRAJ27

>gi 1224589805:22984601-22984666 Homo sapiens chromosome 14, GRCh37 p5

118 Primary Assembly, TRAJ28

>gi 1224589805:22982921-22982980 Homo sapiens chromosome 14, GRCh37 p5

119 Primary Assembly, TRAJ29

>gi 1224589805:22981834-22981890 Homo sapiens chromosome 14, GRCh37 p5

120 Primary Assembly, TRAJ30

>gi 1224589805:22979951-22980007 Homo sapiens chromosome 14, GRCh37 _P5

121 Primary Assembly, TRAJ31

>gi 1224589805:22978325-22978390 Homo sapiens chromosome 14, GRCh37 _P5

122 Primary Assembly, TRAJ32

>gi 1224589805:22977587-22977643 Homo sapiens chromosome 14, GRCh37 _P5

123 Primary Assembly, TRAJ33, tryptophane no phe

>gi 1224589805:22976651-22976708 Homo sapiens chromosome 14, GRCh37 _P5

124 Primary Assembly, TRAJ34

>gi 1224589805:22974096-22974155 Homo sapiens chromosome 14, GRCh37 _P5

125 Primary Assembly, TRAJ36

>gi 1224589805:22972735-22972797 Homo sapiens chromosome 14, GRCh37 _P5

126 Primary Assembly, TRAJ37

>gi 1224589805:22971215-22971276 Homo sapiens chromosome 14, GRCh37 _P5

127 Primary Assembly, TRAJ38, tryptophane no phe

>gi 1224589805:22970585-22970647 Homo sapiens chromosome 14, GRCh37 _P5

128 Primary Assembly, TRAJ39

>gi 1224589805:22968672-22968732 Homo sapiens chromosome 14, GRCh37 _P5

129 Primary Assembly, TRAJ40

>gi 1224589805:22966642-22966703 Homo sapiens chromosome 14, GRCh37 _P5

130 Primary Assembly, TRAJ41

>gi 1224589805:22965872-22965937 Homo sapiens chromosome 14, GRCh37 _P5

131 Primary Assembly, TRAJ42

>gi 1224589805:22964896-22964949 Homo sapiens chromosome 14, GRCh37 _P5

132 Primary Assembly, TRAJ43

>gi 1224589805:22963806-22963868 Homo sapiens chromosome 14, GRCh37 _P5

133 Primary Assembly, TRAJ44

>gi 1224589805:22962911-22962976 Homo sapiens chromosome 14, GRCh37 _P5

134 Primary Assembly, TRAJ45

>gi 1224589805:22962389-22962451 Homo sapiens chromosome 14, GRCh37 _P5

135 Primary Assembly, TRAJ46

>gi 1224589805:22961838-22961894 Homo sapiens chromosome 14, GRCh37 _P5

136 Primary Assembly, TRAJ47

>gi 1224589805:22959479-22959541 Homo sapiens chromosome 14, GRCh37 _P5

137 Primary Assembly, TRAJ48

>gi 1224589805:22958476-22958531 Homo sapiens chromosome 14, GRCh37 _P5

138 Primary Assembly, TRAJ49

>gi 1224589805:22957581-22957640 Homo sapiens chromosome 14, GRCh37 _P5

139 Primary Assembly, TRAJ50

>gi 1224589805:22955216-22955284 Homo sapiens chromosome 14, GRCh37 _P5

140 Primary Assembly, TRAJ52

>gi 1224589805:22951993-22952058 Homo sapiens chromosome 14, GRCh37 _P5

141 Primary Assembly, TRAJ53 >gi 1224589805 : 22951276-22951335 Homo sapiens chromosome 14, GRCh37.p5

142 Primary Assembly, TRAJ54

>gi 1224589805 : 22948510-22948571 Homo sapiens chromosome 14, GRCh37.p5

143 Primary Assembly, TRAJ56

>gi 1224589805 : 22947861-22947923 Homo sapiens chromosome 14, GRCh37.p5

144 Primary Assembly, TRAJ57

145 >TRAV1-1

146 >TRAVl-2

147 >TRAV2

148 >TRAV3

149 >TRAV4

150 >TRAV5

151 >TRAV6

152 >TRAV7

153 >TRAV8-1

154 >TRAV8-2

155 >TRAV8-3

156 >TRAV8-4

157 >TRAV8-6

158 >TRAV9-1

159 >TRAV9-2

160 >TRAV10

161 >TRAV12-1

162 >TRAV12-2

163 >TRAV12-3

164 >TRAV13-1

165 >TRAV13-2

166 >TRAV14/DV4

167 >TRAV16

168 >TRAV17

169 >TRAV18

170 >TRAV19

171 >TRAV20

172 >TRAV21

173 >TRAV22

174 >TRAV23/DV6

175 >TRAV24

176 >TRAV25

177 >TRAV26-1

178 >TRAV26-2

179 >TRAV27

180 >TRAV29/DV5

181 >TRAV30

182 >TRAV34

183 >TRAV35

184 >TRAV36/DV7 185 >TRAV38-1

186 >TRAV38-2/DV8

187 >TRAV39

188 >TRAV40

189 >TRAV41

190 >TRBJ1-1 |gi 189027696: 41921990-41922037

191 >TRBJl-2 |gi 189027696: 41922127-41922174

192 >TRBJl-3 |gi 189027696: 41922740-41922789

193 >TRBJl-4 |gi 189027696: 41923335-41923385

194 >TRBJl-5 |gi 189027696: 41923608-41923657

195 >TRBJl-6 |gi 189027696: 41924098-41924150

196 >TRBJ2-1 |gi 1224589819: 142494049-142494098

197 >TRBJ2-2 |gi 1224589819: 142494244-142494294

198 >TRBJ2-3 |gi | 224589819: 142494531-142494579

199 >TRBJ2-4 |gi 1224589819: 142494682-142494731

200 >TRBJ2-5 |gi 1224589819: 142494803-142494922

201 >TRBJ2-6 |gi | 224589819: 142494895- 142494975

202 >TRBJ2-7 |gi 1224589819: 142495140-142495186

203 >TRBV2 |gi 1224589819: 142000821-142001255

204 >TRBV4-1 |gi 1224589819: 142013036-142013489

205 >TRBV4-2 |gi 1224589819: 142045363-142045816

206 >TRBV4-3 |gi 189027696: 41436393-41436846

207 >TRBV5-1 |gi 1224589819: 142020894-142021363

208 >TRBV5-3 |gi 1224589819 :cl42242747-142242281

209 >TRBV5-4 |gi 1224589819 :cl42168844-142168380

210 >TRBV5-5 |gi 1224589819 :cl42149392-142148928

211 >TRBV5-6 |gi 1224589819 :cl42131877-142131412

212 >TRBV5-7 |gi 1224589819 :cl42111859-142111393

213 >TRBV5-81 gi 189027696 :41635991-41636457

214 >TRBV6-1 |gi 1224589819: 142028178-142028610

215 >TRBV6-2 |gi 189027696: 41422953-41423385

216 >TRBV6-3 |gi 189027696: 41444634-41445066

217 >TRBV6-4 |gi 1224589819 :cl42251137-142250703

218 >TRBV6-5 |gi 1224589819 :cl42180950-142180515

219 >TRBV6-6 |gi 1224589819 :cl42162363-142161931

220 >TRBV6-7 |gi 1224589819 :cl42144084-142143652

221 >TRBV6-8 |gi 1224589819 :cl42124565-142124137

222 >TRBV6-9 |gi 1224589819 :cl42104553-142104121

223 >TRBV7-1 |gi 1224589819: 142032039-142032527

224 >TRBV7-2 |gi 189027696: 41448267-41448763

225 >TRBV7-3 |gi 1224589819 :cl42247565-142247109

226 >TRBV7-4 |gi 1224589819 :cl42176790-142176329

227 >TRBV7-6 |gi 1224589819 :cl42139770-142139278

228 >TRBV7-7 |gi 1224589819 :cl42120321-142119820 229 >TRBV7-8 |gi 1224589819 :cl42099938-142099455

230 >TRBV7-9 |gi 189027696: 41645192-41645664

231 >TRBV9 |gi 1224589819 :cl42240011-142239537

232 >TRBV2 |gi 1224589819: 142000821-142001255

233 >TRBV4-1 gi 1224589819: 142013036-142013489

234 >TRBV4-2 gi 1224589819: 142045363-142045816

235 >TRBV4-3 gi 189027696: 41436393-41436846

236 >TRBV5-1 gi 1224589819: 142020894-142021363

237 >TRBV5-3 gi 1224589819 :cl42242747-142242281

238 >TRBV5-4 gi 1224589819 :cl42168844-142168380

239 >TRBV5-5 gi 1224589819 :cl42149392-142148928

240 >TRBV5-6 gi 1224589819 :cl42131877-142131412

241 >TRBV5-7 gi 1224589819 :cl42111859-142111393

242 >TRBV5-8 gi 189027696 :41635991-41636457

243 >TRBV6-1 gi 1224589819: 142028178-142028610

244 >TRBV6-2 gi 189027696: 41422953-41423385

245 >TRBV6-3 gi 189027696: 41444634-41445066

246 >TRBV6-4 gi 1224589819 :cl42251137-142250703

247 >TRBV6-5 gi 1224589819 :cl42180950-142180515

248 >TRBV6-6 gi 1224589819 :cl42162363-142161931

249 >TRBV6-7 gi 1224589819 :cl42144084-142143652

250 >TRBV6-8 gi 1224589819 :cl42124565-142124137

251 >TRBV6-9 gi 1224589819 :cl42104553-142104121

252 >TRBV7-1 gi 1224589819: 142032039-142032527

253 >TRBV7-2 gi 189027696: 41448267-41448763

254 >TRBV7-3 gi 1224589819 :cl42247565-142247109

255 >TRBV7-4 gi 1224589819 :cl42176790-142176329

256 >TRBV7-6 gi 1224589819 :cl42139770-142139278

257 >TRBV7-7 gi 1224589819 :cl42120321-142119820

258 >TRBV7-8 gi 1224589819 :cl42099938-142099455

259 >TRBV7-9 gi 189027696: 41645192-41645664

260 >TRBV91 gi 224589819 :cl42240011-142239537

261 >TRBV10-1 gi 1224589819 :cl 42232022- 142231573

262 >TRBV10-2 gi 1224589819 :cl 42206960-142206511

263 >TRBV10-3 gi 189027696 :41660123-41660572

264 >TRBV11-1 gi 1224589819 :cl 42224267- 142223790

265 >TRBVll-2 gi 1224589819 :cl 42198008- 142197570

266 >TRBVll-3 gi 189027696 :41670771-41671208

267 >TRBV12-3 gi 189027696 :41676373-41676819

268 >TRBV12-4 gi 189027696 :41679696-41680142

269 >TRBV12-5 gi 189027696 : 41696887-41697333

270 >TRBV13 |gi | 89027696: 41651711-41652194

271 >TRBV141 gi 189027696: 41703830-41704262

272 >TRBV151 gi | 89027696 : 41708905-41709373 273 >TRBV161 gi 189027696: 41713914-41714367

274 >TRBV171 gi 189027696: 41717530-41718264

275 >TRBV181 gi 189027696: 41731601-41732219

276 >TRBV19 |gi 1224589819: 142326571-142327046

277 >TRBV20-1 |gi 1224589819: 142334241-142334913

278 >TRBV21-1 |gi 1224589819: 142344427-142344894

279 >TRBV23-1 |gi 1224589819: 142353468-142353971

280 >TRBV24-1 |gi 1224589819: 142364234-142364710

281 >TRBV25-1 |gi 1224589819: 142378609-142379076

282 >TRBV271 gi 1224589819: 142423216-142423688

283 >TRBV28 |gi 1224589819: 142428504-142428984

284 >TRBV29-1 |gi 1224589819: 142448120-142448741

285 >TRBV301 gi 1224589819 :cl 42510972- 142510271

286 >TRBV13 |gi | 89027696: 41651711-41652194

287 >TRBV141 gi 189027696: 41703830-41704262

288 >TRBV151 gi | 89027696 : 41708905-41709373

289 >TRBV161 gi 189027696: 41713914-41714367

290 >TRBV171 gi 189027696: 41717530-41718264

291 >TRBV181 gi 189027696: 41731601-41732219

292 >TRBV19 |gi 1224589819: 142326571-142327046

293 >TRBV20-1 |gi 1224589819: 142334241-142334913

294 >TRBV21-1 |gi 1224589819: 142344427-142344894

295 >TRBV23-1 |gi 1224589819: 142353468-142353971

296 >TRBV24-1 |gi 1224589819: 142364234-142364710

297 >TRBV25-1 |gi 1224589819: 142378609-142379076

298 >TRBV271 gi 1224589819: 142423216-142423688

299 >TRBV28 |gi 1224589819: 142428504-142428984

300 >TRBV29-1 |gi 1224589819: 142448120-142448741

301 >TRBV301 gi 1224589819 :cl 42510972- 142510271

>gi 1149292731 : cl 14668044- 114667992 Mus musculus strain C57BL/6J

302 chromosome 12, MGSCv37 C57BL/6J, IgHJl

>gi 1149292731 : cl 14667726- 114667679 Mus musculus strain C57BL/6J

303 chromosome 12, MGSCv37 C57BL/6J, IgHJ2

>gi 1149292731 : cl 14667343- 114667296 Mus musculus strain C57BL/6J

304 chromosome 12, MGSCv37 C57BL/6J, IgHJ3

>gi 1149292731 : cl 14666778- 114666725 Mus musculus strain C57BL/6J

305 chromosome 12, MGSCv37 C57BL/6J, IgHJ4

306 >IgHVl-4

307 >IgHVl-5

308 >IgHVl-7

309 >IgHVl-9

310 >IgHVl-ll

311 >IgHVl-12

312 >IgHVl-14

313 >IgHVl-15

314 >IgHVl-17-l 315 >IgHVl-18

316 >IgHVl-19

317 >IgHVl-20

318 >IgHVl-22

319 >IgHVl-26

320 >IgHVl-31

321 >IgHVl-34

322 >IgHVl-36

323 >IgHVl-37

324 >IgHVl-39

325 >IgHVl-42

326 >IgHVl-43

327 >IgHVl-47

328 >IgHVl-49

329 >IgHVl-50

330 >IgHVl-52

331 >IgHVl-53

332 >IgHVl-54

333 >IgHVl-55

334 >IgHVl-56

335 >IgHVl-58

336 >IgHVl-59

337 >IgHVl-61

338 >IgHVl-62-l

339 >IgHVl-62-2

340 >IgHVl-62-3

341 >IgHVl-63

342 >IgHVl-64

343 >IgHVl-66

344 >IgHVl-67

345 >IgHVl-69

346 >IgHVl-71

347 >IgHVl-72

348 >IgHVl-74

349 >IgHVl-75

350 >IgHVl-76

351 >IgHVl-77

352 >IgHVl-78

353 >IgHVl-80

354 >IgHVl-81

355 >IgHVl-82

356 >IgHVl-84

357 >IgHVl-85

358 >IgHV2-2 359 >IgHV2-3

360 >IgHV2-4

361 >IgHV2-5

362 >IgHV2-6

363 >IgHV2-6-8

364 >IgHV2-7

365 >IgHV2-9

366 >IgHV2-9-l

367 >IgHV3-l

368 >IgHV3-2

369 >IgHV3-3

370 >IgHV3-4

371 >IgHV3-5

372 >IgHV3-6

373 >IgHV3-8

374 >IgHV4-l

375 >IgHV4-2

376 >IgHV5-2

377 >IgHV5-4

378 >IgHV5-6

379 >IgHV5-9

380 >IgHV5-9-l

381 >IgHV5-12

382 >IgHV5-12-4

383 >IgHV5-15

384 >IgHV5-16

385 >IgHV5-17

386 >IgHV6-3

387 >IgHV6-4

388 >IgHV6-5

389 >IgHV6-6

390 >IgHV6-7

391 >IgHV7-l

392 >IgHV7-2

393 >IgHV7-3

394 >IgHV7-4

395 >IgHV8-2

396 >IgHV8-4

397 >IgHV8-5

398 >IgHV8-6

399 >IgHV8-8

400 >IgHV8-ll

401 >IgHV8-12

402 >IgHV9-l 403 >IgHV9-2

404 >IgHV9-3

405 >IgHV9-4

406 >IgHV10-l

407 >IgHV10-3

408 >IgHVll-l

409 >IgHVll-2

410 >IgHV12-3

411 >IgHV13-l

412 >IgHV13-2

413 >IgHV14-l

414 >IgHV14-2

415 >IgHV14-3

416 >IgHV14-4

417 >IgHV15-2

418 >IgHV16-l

>gi 149292735:54837511-54837576 Mus musculus strain C57BL/6J chromosome

419 14, MGSCv37 C57BL/6J, TRAJ2

>gi 149292735:54833452-54833513 Mus musculus strain C57BL/6J chromosome

420 14, MGSCv37 C57BL/6J, TRAJ5

>gi 149292735:54832363-54832424 Mus musculus strain C57BL/6J chromosome

421 14, MGSCv37 C57BL/6J, TRAJ6

>gi 149292735:54829068-54829125 Mus musculus strain C57BL/6J chromosome

422 14, MGSCv37 C57BL/6J, TRAJ9

>gi 149292735:54826814-54826872 Mus musculus strain C57BL/6J chromosome

423 14, MGSCv37 C57BL/6J, TRAJ11

>gi 149292735:54826227-54826285 Mus musculus strain C57BL/6J chromosome

424 14, MGSCv37 C57BL/6J, TRAJ12

>gi 149292735:54825416-54825472 Mus musculus strain C57BL/6J chromosome

425 14, MGSCv37 C57BL/6J, TRAJ13

>gi 149292735:54824097-54824156 Mus musculus strain C57BL/6J chromosome

426 14, MGSCv37 C57BL/6J, TRAJ15

>gi 149292735:54822809-54822869 Mus musculus strain C57BL/6J chromosome

427 14, MGSCv37 C57BL/6J, TRAJ16

>gi 149292735:54821450-54821512 Mus musculus strain C57BL/6J chromosome

428 14, MGSCv37 C57BL/6J, TRAJ17

>gi 149292735:54820452-54820517 Mus musculus strain C57BL/6J chromosome

429 14, MGSCv37 C57BL/6J, TRAJ18

>gi 149292735:54818465-54818521 Mus musculus strain C57BL/6J chromosome

430 14, MGSCv37 C57BL/6J, TRAJ21

>gi 149292735:54816923-54816983 Mus musculus strain C57BL/6J chromosome

431 14, MGSCv37 C57BL/6J, TRAJ22

>gi 149292735:54815756-54815815 Mus musculus strain C57BL/6J chromosome

432 14, MGSCv37 C57BL/6J, TRAJ23

>gi 149292735:54815320-54815375 Mus musculus strain C57BL/6J chromosome

433 14, MGSCv37 C57BL/6J, TRAJ24

>gi 149292735:54814165-54814224 Mus musculus strain C57BL/6J chromosome

434 14, MGSCv37 C57BL/6J, TRAJ26

>gi 149292735:54811978-54812036 Mus musculus strain C57BL/6J chromosome

435 14, MGSCv37 C57BL/6J, TRAJ27

>gi 149292735:54811336-54811400 Mus musculus strain C57BL/6J chromosome

436 14, MGSCv37 C57BL/6J, TRAJ28

>gi 149292735:54809541-54809599 Mus musculus strain C57BL/6J chromosome

437 14, MGSCv37 C57BL/6J, TRAJ30 >gi 149292735:54807570-54807626 Mus musculus strain C57BL/6J chromosome

438 14, MGSCv37 C57BL/6J, TRAJ31

>gi 149292735:54805776-54805841 Mus musculus strain C57BL/6J chromosome

439 14, MGSCv37 C57BL/6J, TRAJ32

>gi 149292735:54805033-54805089 Mus musculus strain C57BL/6J chromosome

440 14, MGSCv37 C57BL/6J, TRAJ33, tryptophane no phe

>gi 149292735:54804374-54804431 Mus musculus strain C57BL/6J chromosome

441 14, MGSCv37 C57BL/6J, TRAJ34

>gi 149292735:54803449-54803513 Mus musculus strain C57BL/6J chromosome

442 14, MGSCv37 C57BL/6J, TRAJ35

>gi 149292735:54801193-54801252 Mus musculus strain C57BL/6J chromosome

443 14, MGSCv37 C57BL/6J, TRAJ37

>gi 149292735:54799637-54799699 Mus musculus strain C57BL/6J chromosome

444 14, MGSCv37 C57BL/6J, TRAJ39

>gi 149292735:54797596-54797656 Mus musculus strain C57BL/6J chromosome

445 14, MGSCv37 C57BL/6J, TRAJ40

>gi 149292735:54795448-54796511 Mus musculus strain C57BL/6J chromosome

446 14, MGSCv37 C57BL/6J, TRAJ42

>gi 149292735:54794417-54794473 Mus musculus strain C57BL/6J chromosome

447 14, MGSCv37 C57BL/6J, TRAJ43

>gi 149292735:54792506-54792565 Mus musculus strain C57BL/6J chromosome

448 14, MGSCv37 C57BL/6J, TRAJ45

>gi 149292735:54789483-54789543 Mus musculus strain C57BL/6J chromosome

449 14, MGSCv37 C57BL/6J, TRAJ48

>gi 149292735:54788361-54788419 Mus musculus strain C57BL/6J chromosome

450 14, MGSCv37 C57BL/6J, TRAJ49

>gi 149292735:54787265-54787327 Mus musculus strain C57BL/6J chromosome

451 14, MGSCv37 C57BL/6J, TRAJ50

>gi 149292735:54784991-54785056 Mus musculus strain C57BL/6J chromosome

452 14, MGSCv37 C57BL/6J, TRAJ52

>gi 149292735:54782319-54782384 Mus musculus strain C57BL/6J chromosome

453 14, MGSCv37 C57BL/6J, TRAJ53

>gi 149292735:54778938-54779000 Mus musculus strain C57BL/6J chromosome

454 14, MGSCv37 C57BL/6J, TRAJ56

>gi 149292735:54778182-54778244 Mus musculus strain C57BL/6J chromosome

455 14, MGSCv37 C57BL/6J, TRAJ57

>gi 149292735:54776955-54777017 Mus musculus strain C57BL/6J chromosome

456 14, MGSCv37 C57BL/6J, TRAJ58

457 >TRAV1

458 >TRAV2

459 >TRAV3-1

460 >TRAV3-3

461 >TRAV3-4

462 >TRAV3D-3

463 >TRAV3N-3

464 >TRAV4-2

465 >TRAV4-3

466 >TRAV4-4/DV10

467 >TRAV4D-3

468 >TRAV4D-4

469 >TRAV4N-3

470 >TRAV4N-4

471 >TRAV5-1

472 >TRAV5D-4 473 >TRAV5N-4

474 >TRAV6-1

475 >TRAV6-2

476 >TRAV6-3

477 >TRAV6-4

478 >TRAV6-5

479 >TRAV6-6

480 >TRAV6-7/DV9

481 >TRAV6D-3

482 >TRAV6D-4

483 >TRAV6D-5

484 >TRAV6D-6

485 >TRAV6D-7

486 >TRAV6N-5

487 >TRAV6N-6

488 >TRAV6N-7

489 >TRAV7-1

490 >TRAV7-2

491 >TRAV7-3

492 >TRAV7-4

493 >TRAV7-5

494 >TRAV7-6

495 >TRAV7D-2

496 >TRAV7D-3

497 >TRAV7D-4

498 >TRAV7D-5

499 >TRAV7D-6

500 >TRAV7N-4

501 >TRAV7N-6

502 >TRAV8-1

503 >TRAV8-2

504 >TRAV8D-1

505 >TRAV8D-2

506 >TRAV8N-2

507 >TRAV9-1

508 >TRAV9-2

509 >TRAV9-3

510 >TRAV9-4

511 >TRAV9D-1

512 >TRAV9D-2

513 >TRAV9D-3

514 >TRAV9N-2

515 >TRAV9N-4

516 >TRAV10 517 >TRAV10D

518 >TRAV10N

519 >TRAV11

520 >TRA11-D

521 >TRAV12-1

522 >TRAV12-2

523 >TRAV12-3

524 >TRAV12D-1

525 >TRAV12D-2

526 >TRAV12D-3

527 >TRAV12N-1

528 >TRAV12N-2

529 >TRAV12N-3

530 >TRAV13-1

531 >TRAV13-2

532 >TRAV13-3

533 >TRAV13-4/DV7

534 >TRAV13-5

535 >TRAV13D-1

536 >TRAV13D-2

537 >TRAV13D-3

538 >TRAV13D-4

539 >TRAV13N-1

540 >TRAV13N-2

541 >TRAV13N-3

542 >TRAV13N-4

543 >TRAV14-1

544 >TRAV14-2

545 >TRAV14-3

546 >TRAV14D-1

547 >TRAV14D-2

548 >TRAV14D-3/DV8

549 >TRAV14N-1

550 >TRAV14N-2

551 >TRAV14N-3

552 >TRAV15-1/DV6-1

553 >TRAV15-2/DV6-2

554 >TRAV15D-1/DV6D-1

555 >TRAV15D-2/DV6D-3

556 >TRAV15N-1

557 >TRAV15N-2

558 >TRAV16

559 >TRAV16D/DV11

560 >TRAV16N 561 >TRAV17

562 >TRAV19

563 >TRAV21/DV12

564 >TRBJ1-1

565 >TRBJl-2

566 >TRBJl-3

567 >TRBJl-4

568 >TRBJl-5

569 >TRBJ2-1

570 >TRBJ2-2

571 >TRBJ2-3

572 >TRBJ2-4

573 >TRBJ2-5

574 >TRBJ2-7

575 >TRBV1

576 >TRBV2

577 >TRBV3

578 >TRBV4

579 >TRBV5

580 >TRBV12-1

581 >TRBV12-2

582 >TRBV13-1

583 >TRBV13-2

584 >TRBV13-3A

585 >TRBV14

586 >TRBV15

587 >TRBV16

588 >TRBV17

589 >TRBV19

590 >TRBV20

591 >TRBV23

592 >TRBV26

593 >TRBV29

594 >TRBV30

595 >TRBV31

Claims

CLAIMS What is claimed is:

1. A method for immune repertoire sequence identification which comprises:

a. comparing an unknown sequence to a plurality of immune repertoire specific

reference sequences ("words");

b. if a portion of the unknown sequence matches an immune repertoire reference

sequence, measuring a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence; and

c. using the measured distance and the immune repertoire reference sequence to identify the unknown sequence.

2. The method of Claim 1, wherein multiplexed polymerase chain reaction is used to amplify the unknown sequence.

3. The method of Claim 1, wherein massively parallel sequencing is used to sequence the unknown sequence.

4. The method of Claim 1, wherein the immune repertoire specific reference sequences are immunoglobulin IgH or immunoglobulin IgL reference sequences.

5. The method of Claim 1, wherein the immune repertoire specific reference sequences are T cell receptor (TCR ) or T cell receptor (TCRoc) reference sequences.

6. The method of Claim 1, wherein the immune repertoire specific reference sequences are joining (J) gene reference sequences.

7. The method of Claim 1, wherein the immune repertoire specific reference sequences are variable (V) gene reference sequences.

8. The method of Claim 1, wherein the conserved reference codon is a second conserved cysteine, a phenylalanine or a tryptophan codon.

9. The method of Claim 1, wherein the immune repertoire specific reference sequences are selected from the sequences in Table 1.

10. The method of Claim 1, wherein the conserved reference codon is a codon selected from the reference codons in Table 1.

11. The method of Claim 1, wherein the distance to the conserved reference codon is a distance selected from the distances to the reference codons in Table 1.

12. A computer-implemented method for immune repertoire sequence identification which comprises: comparing an unknown sequence to a plurality of immune repertoire specific reference sequences ("words");

if a portion of the unknown sequence matches an immune repertoire reference sequence, measuring a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence; and

using the measured distance and the immune repertoire reference sequence to identify the unknown sequence.

A system for immune repertoire sequence identification which comprises:

an alignment module wherein an unknown sequence is aligned with a plurality of immune repertoire specific reference sequences ("words"); and

a measurement module that measures and compares a distance from the immune repertoire reference sequence to a conserved reference codon in the unknown sequence.