DIAGNOSTIC METHODS USING HIGH-THROUGHPUT TCRß SEQUENCING AND USES THEREOF

Abstract

A method for diagnosing or monitoring an autoimmune disease, an immunity response post-vaccination, or a post-stem cell transplant reaction in a subject which includes high throughput T-Cell Receptor β (TCRβ) sequencing tests to identify dominant clone(s) in a subject suffering from the autoimmune disease, or in a subject post-vaccination administration, or in a subject post-stem cell transplantation. Oligonucleotides primer compositions and kits associated with the above-described methods are also provided herein. Said subject includes living organisms, such as mammals (e.g., dogs, cats, pigs, cows, horses, goats, rabbits, humans), non-mammalian vertebrates, such as birds (e.g., chicken, ducks), fish (e.g., sharks), or frogs, and transgenic species thereof.

1 .- 25 . (canceled) 
     
     
         26 . A method for comprising the steps of:
 (i) creating a T cell receptor β chain (TCRβ) DNA library using a biological sample obtained from a subject,   (ii) assessing the frequency of each TCR clonotype;   (iii) identifying an autoimmune syndrome TCR clonotype using one or more clonal abundance indicies;   (iv) comparing the frequency of the autoimmune syndrome TCR clonotype to an abundance threshold criterion derived from the frequency of the dominant clone in samples obtained from a plurality of subjects diagnosed as not having the autoimmune disease; and   (v) identifying the subject as having the autoimmune disease when the frequency is above the abundance threshold criterion on identifying the subject as not having the autoimmune disease when the frequency is below the abundance threshold criterion,   wherein the creation of the TCRβ DNA library comprises the steps of:   (a) isolating RNA from the biological sample,   (b) generating DNA product via reverse transcription of the isolated RNA and amplifying the resultant DNA product,   (c) analyzing the amplified DNA product, and   (d) sequencing of the amplified DNA product; and   wherein the step of amplification of the resultant DNA product comprises incorporating a randomized molecular tag that is contiguous with an adaptor sequence into each molecule of input TCR, and wherein the incorporation of the randomized molecular tag that is contiguous with the adaptor sequence comprises the step of annealing each molecule of input TCR mRNA to a primer having the randomized molecular tag that is contiguous with the adaptor sequence.   
     
     
         27 . A method for comprising the steps of:
 (i) creating a T cell receptor β chain (TCRβ) DNA library using a biological sample obtained from a subject,   (ii) assessing the frequency of each TCR clonotype;   (iii) identifying a post-vaccination response TCR clonotype using one or more clonal abundance indicies;   (iv) comparing the frequency of the post-vaccination response TCR clonotype to an abundance threshold criterion derived from the frequency of the dominant clone in samples obtained from a plurality of subjects diagnosed as not having a post-vaccination response; and   (v) identifying the subject as having the post-vaccination response when the frequency is above the abundance threshold criterion on identifying the subject as not having the post-vaccination response when the frequency is below the abundance threshold criterion,   wherein the creation of the TCRβ DNA library comprises the steps of:   (a) isolating RNA from the biological sample,   (b) generating DNA product via reverse transcription of the isolated RNA and amplifying the resultant DNA product,   (c) analyzing the amplified DNA product, and   (d) sequencing of the amplified DNA product; and   wherein the step of amplification of the resultant DNA product comprises incorporating a randomized molecular tag that is contiguous with an adaptor sequence into each molecule of input TCR, and wherein the incorporation of the randomized molecular tag that is contiguous with the adaptor sequence comprises the step of annealing each molecule of input TCR mRNA to a primer having the randomized molecular tag that is contiguous with the adaptor sequence.   
     
     
         28 . The method of  claim 27 , wherein the post-vaccination response is an increased number of healthy T-cells in the subject relative to a number of healthy T-cell in the subject pre-vaccination. 
     
     
         29 . A method for comprising the steps of:
 (i) creating a T cell receptor β chain (TCRβ) DNA library using a biological sample obtained from a subject,   (ii) assessing the frequency of each TCR clonotype;   (iii) identifying a post-stem cell transplantation response TCR clonotype using one or more clonal abundance indicies;   (iv) comparing the frequency of the post-stem cell transplantation response TCR clonotype to an abundance threshold criterion derived from the frequency of the dominant clone in samples obtained from a plurality of subjects diagnosed as not having the post-stem cell transplantation response; and   (v) identifying the subject as having the post-stem cell transplantation response when the frequency is above the abundance threshold criterion on identifying the subject as not having the post-stem cell transplantation response when the frequency is below the abundance threshold criterion,   wherein the creation of the TCRβ DNA library comprises the steps of:   (a) isolating RNA from the biological sample,   (b) generating DNA product via reverse transcription of the isolated RNA and amplifying the resultant DNA product,   (c) analyzing the amplified DNA product, and   (d) sequencing of the amplified DNA product; and   wherein the step of amplification of the resultant DNA product comprises incorporating a randomized molecular tag that is contiguous with an adaptor sequence into each molecule of input TCR, and wherein the incorporation of the randomized molecular tag that is contiguous with the adaptor sequence comprises the step of annealing each molecule of input TCR mRNA to a primer having the randomized molecular tag that is contiguous with the adaptor sequence.   
     
     
         30 . The method of  claim 29 , wherein the post-stem cell transplantation response is an increased number of T-cells in the subject relative to a number of T-cells pre-stem cell transplantation in the subject. 
     
     
         31 . The method of  claim 26 , wherein the biological sample is lymph node tissue, peripheral blood lymphocytes, bone marrow, gut associated lymphoid tissue, other tissue having a malignant lymphoid lesion, or a combination thereof. 
     
     
         32 . The method of  claim 26 , wherein the samples are lymph node tissue, peripheral blood lymphocytes, bone marrow, gut associated lymphoid tissue, other tissue having a malignant lymphoid lesion, or a combination thereof. 
     
     
         33 . The method of  claim 26 , wherein the steps of reverse transcription and amplification are performed using RT-PCR. 
     
     
         34 . The method of  claim 26 , wherein the step of sequencing of the amplified DNA product is performed using high-throughput sequencing. 
     
     
         35 . The method of  claim 26 , wherein the abundance threshold criterion is greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, or 15%. 
     
     
         36 . The method of  claim 26 , wherein the abundance threshold criterion is greater than or equal to 5%. 
     
     
         37 . The method of  claim 26 , wherein the primer has a nucleic acid sequence comprising one or more of SEQ ID NO. 5 through SEQ ID NO. 26. 
     
     
         38 . The method of  claim 26 , wherein the primer has a nucleic acid sequence comprising one or more of SEQ ID NO. 17 through SEQ ID NO. 26. 
     
     
         39 . The method of  claim 26 , wherein the primer has a nucleic acid sequence comprising SEQ ID NO. 5 through SEQ ID NO. 26. 
     
     
         40 . A kit for diagnosing a subject suspected to be suffering from or susceptible to an autoimmune disease, the kit comprising at least one forward primer and at least one reverse primer, the forward primer and the reverse primer each has a nucleic acid sequence comprising one or more of SEQ ID NO. 5 through SEQ ID NO. 26, together with instructions for carrying out the method of  claim 26 . 
     
     
         41 . A kit for diagnosing a subject suspected to be suffering from or susceptible to an autoimmune disease, the kit comprising at least one forward primer and at least one reverse primer, the forward primer and the reverse primer each has a nucleic acid sequence comprising one or more of SEQ ID NO. 17 through SEQ ID NO. 26, together with instructions for carrying out the method of  claim 26 . 
     
     
         42 . A kit for diagnosing a subject suspected to be suffering from or susceptible to an autoimmune disease, the kit comprising at least one forward primer and at least one reverse primer, the forward primer and the reverse primer each has a nucleic acid sequence comprising SEQ ID NO. 5 through SEQ ID NO. 26, together with instructions for carrying out the method of  claim 28 . 
     
     
         43 . The method of  claim 26 , wherein the subject is canine. 
     
     
         44 . The method of  claim 26 , wherein the randomized molecular tag is a random sequence of contiguous nucleotides selected from A, C, T or G, and is less than 10 nucleotides in length. 
     
     
         45 . The method of  claim 26 , wherein the adaptor sequence comprises at least one of SEQ ID NO: 49 through SEQ ID NO: 56.