Abstract: Methods and compositions are provided for quantifying the number of biological input molecules of one or more target genes of interest in a PCR. The method comprises steps for amplifying multiple synthetic templates and high throughput sequencing. The method further comprises steps for achieving equal sequencing coverage between targets that do not occur in equal ratios, while mitigating the need for costly deep sequencing methods.

Abstract: Methods are provided for correction of amplification bias and quantitation of adaptive immune cells in a sample using synthetic templates that include random oligonucleotide sequences.

Abstract: The present disclosure provides TCRs with high or enhanced affinity against various tumor associated antigens (including human Wilms tumor protein 1 (WT 1) epitopes and mesothelin epitopes), T cells expressing such high affinity antigen specific TCRs, nucleic acids encoding the same, and compositions for use in treating diseases or disorders in which cells overexpress one or more of these antigens, such as in cancer.

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 template 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: Methods are provided for correction of amplification bias and quantitation of adaptive immune cells in a sample using synthetic templates that include random oligonucleotide sequences.

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

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: The present disclosure provides TCRs with high or enhanced affinity against various tumor associated antigens (including human Wilms tumor protein 1 (WT 1) epitopes and mesothelin epitopes), T cells expressing such high affinity antigen specific TCRs, nucleic acids encoding the same, and compositions for use in treating diseases or disorders in which cells overexpress one or more of these antigens, such as in cancer.

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: 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: 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: Methods are provided for correction of amplification bias and quantitation of adaptive immune cells in a sample using synthetic templates that include random oligonucleotide sequences.

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: 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: 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: 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: 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 N 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: 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.