Abstract: The invention is directed to sequencing-based methods for monitoring a minimal residual disease of a diffuse large B cell lymphoma (DLBCL) by one or more clonotypes correlated with the disorder. In some embodiments, such methods comprise the following steps: (a) obtaining a sample of peripheral blood from the patient; (b) amplifying molecules of nucleic acid from the sample, the molecules of nucleic acid comprising recombined DNA sequences from immunoglobulin genes; (c) sequencing the amplified molecules of nucleic acid to form a clonotype profile; and (d) determining from the clonotype profile a presence, absence and/or level of one or more patient-specific clonotypes correlated with the DLBCL and phylogenic clonotypes thereof.

Abstract: The invention is directed to methods for determining antigen-specific T cells. In some embodiments, methods of the invention may be implemented by the steps of reacting under interaction conditions one or more antigens with T cells in a plurality of subsets of a tissue sample, such as peripheral blood; sorting antigen-interacting T cells from other T cells; separately sequencing for each subset recombined nucleic acid encoding a segment of a TCR chain from a sample of T cells prior to exposure to antigen and from a sample of T cells isolated based on their interaction with antigen, thereby forming a clonotype profile for the former sample and the latter sample for each subset; and identifying as antigen-specific T cells those T cells associated with a clonotype whose frequency increases in the latter sample relative to its frequency in the former sample.

Abstract: The invention is directed to methods for determining clonotypes and clonotype profiles in assays for analyzing immune repertoires by high throughput nucleic acid sequencing of somatically recombined immune molecules. In one aspect, the invention comprises generating a clonotype profile from an individual by generating sequence reads from a sample of recombined immune molecules; forming from the sequence reads a sequence tree representing candidate clonotypes each having a frequency; coalescing with a highest frequency candidate clonotype any lesser frequency candidate clonotypes whenever such lesser frequency is below a predetermined value and whenever a sequence difference therebetween is below a predetermined value to form a clonotype. After such coalescence, the candidate clonotypes is removed from the sequence tree and the process is repeated.

Abstract: The invention is directed to methods for determining clonotypes and clonotype profiles in assays for analyzing immune repertoires by high throughput nucleic acid sequencing of somatically recombined immune molecules. In one aspect, the invention comprises generating a clonotype profile from an individual by generating sequence reads from a sample of recombined immune molecules; forming from the sequence reads a sequence tree representing candidate clonotypes each having a frequency; coalescing with a highest frequency candidate clonotype any lesser frequency candidate clonotypes whenever such lesser frequency is below a predetermined value and whenever a sequence difference therebetween is below a predetermined value to form a clonotype. After such coalescence, the candidate clonotypes is removed from the sequence tree and the process is repeated.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer, especially lymphoid neoplasms, such as lymphomas and leukemias. Provided herein are methods for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions. Identified biomarkers can be used to determine and/or monitor the disease state for a subject with an associated lymphoid disorder or autoimmune disease or other condition. In particular, the invention provides a sensitive method for monitoring lymphoid neoplasms that undergo clonal evolutions without the need to development alternative assays for the evolved or mutated clones serving as patient-specific biomarkers.

Abstract: The invention is directed to methods of measuring an immune response by comparing sequence-based clonotype frequency data from successively measured clonotype profiles. In particular, the invention includes immunotherapies of cancers, such as lymphomas, that include sensitive pre- and post-vaccination sequence-based measurements of changes in a patient's immune repertoire, thereby providing a sensitive measure of the likelihood of treatment success.

Abstract: The invention is directed to a method of prognosing in an individual a transformation from follicular lymphoma to diffuse large B-cell lymphoma (DLBCL) by measuring changes and/or lack of changes in certain groups of related clonotypes, referred to herein as “clans,” in successive clonotype profiles of the individual. A clan may arise from a single lymphocyte progenitor that gives rise to many related lymphocyte progeny, each possessing and/or expressing a slightly different immunoglobulin receptor due to somatic mutation(s), such as base substitutions, inversions, related rearrangements resulting in common V(D)J gene segment usage, or the like. A higher likelihood of transformation from follicular lymphoma to DLBCL is correlated with the persistence of clans in successive clonotype profiles whose clonotype membership fails to undergo diversification over time.

Abstract: The invention is directed to methods of generating sequence profiles of populations of nucleic acids, whose member nucleic acids contain regions of high variability, such as populations of nucleic acids encoding T cell receptors or B cell receptors. In one aspect, the invention provides pluralities of sets of primers for generating nested sets of templates from nucleic acids in such populations, thereby insuring the production of at least one template from which sequence reads are generated, despite such variability, or despite limited lengths or quality of sequence reads. In another aspect, members of such populations are bidirectionally sequenced so that further sequence information is obtained by analyzing overlapping sequence reads in the zones of highest variability.

Abstract: The invention is directed to a method of monitoring or detecting treatment-resistant clones in a patient being treated for a lymphoid or myeloid neoplasm from which patient-specific correlating clonotypes have been identified. In some embodiments, such method includes the steps of obtaining a sample from the patient comprising T-cells and/or B-cells; amplifying molecules of nucleic acid from the T-cells and/or B-cells of the sample, the molecules of nucleic acid comprising recombined DNA sequences from T-cell receptor genes or immunoglobulin genes; sequencing the amplified molecules of nucleic acid to form a clonotype profile; determining from the clonotype profile a level of each correlating clonotype and clonotypes clonally evolved therefrom; and correlating a presence of a treatment-resistant clone of the neoplasm with a change in relative levels of the correlating clonotypes and clonotypes clonally evolved therefrom.

Abstract: The invention relates to methods of labeling nucleic acids, such as fragments of genomic DNA, with unique sequence it referred to herein as “mosaic tag,” prior to amplification and/or sequencing. Such sequence tags are useful for identifying amplification and sequencing errors. Mosaic tags minimize sequencing and amplification artifacts due to inappropriate annealing priming, hairpin formation, or the like, that may occur with completely random sequence tags of the prior art. In one aspect, mosaic tags are sequence tags that comprise alternating constant regions and variable regions, wherein each constant region has it position in the mosaic tag and comprises a predetermined sequence of nucleotides and each variable region has a position in the mosaic tag and comprises a predetermined number of randomly selected nucleotides.

Abstract: The invention is directed to a method of selecting disease-correlated clonotypes that have a reduced likelihood of producing a false positive signal of relapse when used to monitor minimal residual disease. In accordance with the invention, candidate correlating clonotypes are obtained from a patient, the rarity of each is determined either by comparison with a clonotype database or a clonotype model, and one or more of the rarest of such clonotypes are used to monitor the minimal residual disease.

Abstract: There is a need or improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer, especially lymphoid neoplasms, such as lymphomas and leukemias. Provided herein are methods for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions. Identified biomarkers can be used to determine and/or monitor the disease state for a subject with an associated lymphoid disorder or autoimmune disease or other condition. In particular, the invention provides a sensitive method for monitoring lymphoid neoplasms that undergo clonal evolutions without the need to development alternative assays for the evolved or mutated clones serving as patient-specific biomarkers.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer. Provided herein are methods for using DNA sequencing to identify personalized biomarkers in patients with autoimmune disease and other conditions. Identified biomarkers can be used to determine the disease state for a subject with an autoimmune disease or other condition.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer. Provided herein are methods for using DNA sequencing to identify personalized biomarkers in patients with autoimmune disease and other conditions. Identified biomarkers can be used to determine the disease state for a subject with an autoimmune disease or other condition.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer, especially lymphoid neoplasms, such as lymphomas and leukemias. Provided herein are methods for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions. Identified biomarkers can be used to determine and/or monitor the disease state for a subject with an associated lymphoid disorder or autoimmune disease or other condition. In particular, the invention provides a sensitive method for monitoring lymphoid neoplasms that undergo clonal evolutions without the need to development alternative assays for the evolved or mutated clones serving as patient-specific biomarkers.

Abstract: The invention is directed to methods of generating sequence profiles of populations of nucleic acids, whose member nucleic acids contain regions of high variability, such as populations of nucleic acids encoding T cell receptors or B cell receptors. In one aspect, the invention provides pluralities of sets of primers for generating nested sets of templates from nucleic acids in such populations, thereby insuring the production of at least one template from which sequence reads are generated, despite such variability, or dispite limited lengths or quality of sequence reads. In another aspect, members of such populations are bidirectionally sequenced so that further sequence information is obtained by analyzing overlapping sequence reads in the zones of highest variability.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer, especially lymphoid neoplasms, such as lymphomas and leukemias. Provided herein are methods for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions. Identified biomarkers can be used to determine and/or monitor the disease state for a subject with an associated lymphoid disorder or autoimmune disease or other condition. In particular, the invention provides a sensitive method for monitoring lymphoid neoplasms that undergo clonal evolutions without the need to development alternative assays for the evolved or mutated clones serving as patient-specific biomarkers.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer. Provided herein are methods for using DNA sequencing to identify personalized biomarkers in patients with autoimmune disease and other conditions. Identified biomarkers can be used to determine the disease state for a subject with an autoimmune disease or other condition.

Abstract: The invention is directed to a method for determining immunophenotypes of tissue-infiltrating lymphocytes in a solid tissue of a patient by (a) generating clonotype profiles from a sample of nucleic acid extracted from a fixed tissue sample from a solid tissue of the patient, where such tissue contains tissue-infiltrating lymphocytes; and (b) determining immunophenotypes of the tissue-infiltrating lymphocytes by (i) obtaining a sample of lymphocytes from peripheral blood of the patient; (ii) sorting the lymphocytes from peripheral blood into at least one subset based on different immunophenotypes of the lymphocytes; (iii) generating a clonotype profile for each of the at least one subset of lymphocytes; and (iv) determining immunophenotypes of lymphocytes in the fixed tissue sample by a correspondence between clonotypes of the fixed tissue sample and clonotypes of the at least one subset.

Abstract: The invention is directed to the use of sequence tags to improve sequence determination of amplicons of related sequences, particularly large and complex amplicons, such as those comprising recombined nucleic acids encoding immune receptor molecules. In one aspect, sequence reads having the same sequence tags are aligned after which final base calls are determined from a (possibly weighted) average base call from sequence read base calls at each position. Similarly, in another aspect, sequence reads comprising series of incorporation signals are aligned by common sequence tags and base calls in homopolymer regions are made as a function incorporation signal values at each “flow” position.