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 highly-multiplexed simultaneous detection of nucleic acids encoding paired adaptive immune heterodimers from a large number of biological samples containing lymphocytes of interest. Methods of the invention comprise performing a single pairing assay on a pool of source samples to determine nucleic acids encoding paired cognate receptor heterodimer chains in the combined pool. Separately, single-locus, high-throughput sequencing is performed on each individual sample to determine the plurality of sequences encoding one of the two receptor polypeptide chains. Pairs of cognate sequences determined in the pooled sample may then be mapped back to a single source sample by comparing the expression patterns of the single-locus sequences.

Abstract: The invention is directed to a method of sequencing low-complexity amplicons randomly arrayed at high density on a surface. Methods of the invention include preparing amplicons for sequencing by a sets of primers that ensure initial signals front different amplicons on the surface will be evenly distributed among the different nucleotides being added in a sequencing by synthesis operation.

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 relates to methods and compositions for estimating the absolute abundance individually for each unique rearranged lymphocyte receptor in a mixed sample.

Abstract: Disclosed are methods for determining the immunological status of the adaptive immune system of a subject by identifying and quantifying rearranged DNA (and/or subsequently transcribed RNA) sequences encoding T cell receptor (TCR) and/or immunoglobulin (IG) polypeptides, in a lymphoid DNA-containing sample from the subject TCR and/or IG sequence diversity and sequence distribution permit immunocompetence and immune repertoire assessment and reflect the degree of T cell or B cell clonality and clerical expansion in the sample. Methods for stratifying patient populations on the basis of immunocompetence including likelihood of responding to immunotherapy are also described.

Abstract: Compositions and methods are described for standardizing the DNA amplification efficiencies of a highly heterogeneous set of oligonucleotide primers as may typically be used to amplify a heterogeneous set of DNA templates that contains rearranged lymphoid cell DNA encoding T cell receptors (TCR) or immunoglobulins (IG). The presently disclosed embodiments are useful to overcome undesirable bias in the utilization of a subset of amplification primers, which leads to imprecision in multiplexed high throughput sequencing of amplification products to quantify unique TCR or Ig encoding genomes in a sample. Provided is a composition comprising a diverse plurality of template oligonucleotides in substantially equimolar amounts, for use as a calibration standard for amplification primer sets. Also provided are methods for identifying and correcting biased primer efficiency during amplification.

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: A relative representation of adaptive immune cells in a biological sample is quantified using multiplex PCR and sequencing of adaptive immune cells, control genes, and synthetic template molecules. Disclosed herein are methods for quantifying a number of adaptive immune cells in a biological sample, and methods for quantifying a relative representation of adaptive immune cells in a biological sample that comprises a mixture of cells comprising adaptive immune cells and cells that are not adaptive immune cells. Methods are provided for amplifying by multiplex PCR and sequencing a first set of synthetic templates each comprising one TCR or IgV segment and one TCR of Ig J or C segment and a unique bar code.

Abstract: Methods are described for diagnosis of a lymphoid hematological malignancy in a subject prior to treatment, and for detecting minimal residual disease (MRD) in the subject after treatment for the malignancy, by high throughput quantitative sequencing (HTS) of multiple unique adaptive immune receptor (TCR or Ig) encoding DNA molecules that have been amplified from DNA isolated from blood samples or other lymphoid cell-containing samples. Amplification employs oligonucleotide primer sets designed to amplify CDR3-encoding sequences within substantially all possible human VDJ or VJ combinations. Disease-characteristic adaptive immune receptor clonotypes occur, prior to treatment, at a relative frequency of at least 15-30% of rearranged receptor CDR3-encoding gene regions. Following treatment, persistence of at least one such clonotype at a detectable frequency of at least 10?6 or at least 10?5 receptor CDR3-encoding regions indicates MRD.

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 present invention is drawn to methods for measuring numbers, levels, and/or ratios of cells, such as lymphocytes, infiltrated into a solid tissue, such as a tumor or a tissue affected by an autoimmune disease, and to methods for making patient prognoses based on such measurements. In one aspect, methods of the invention comprise sorting lymphocytes from an accessible tissue, such as peripheral blood, into functional subsets, such as cytotoxic T cells and regulatory T cells, and generating clonotype profiles of each subset. An inaccessible disease-affected tissue is sampled and one or more clonotype profiles are generated. From the latter clonotype profiles, levels lymphocytes in each of the functional subsets are determined in the disease-affected tissue by their clonotypes, which are identified from lymphocytes sorted into subsets from the accessible tissue.

Abstract: The invention provides a method of making measurements on individual cells of a population by forming reactors containing single cells and a predetermined number, usually one, homogeneous sequence tag. In one aspect, the invention provides a method of making multiparameter measurements on individual cells of such a population by carrying out a polymerase cycling assembly (PCA) reaction to link their identifying nucleic acid sequences, such as sequence tag copies derived from a homogeneous sequence tag, to other cellular nucleic acids of interest, thereby forming fusion products. The fusion products of such PCA reactions are then sequenced and tabulated to generate multiparameter data for cells of the population.