Abstract: Provided herein are methods of determining that a subject has or is at risk of having a disease (e.g., cancer) using analysis of gene expression of loci identified in nucleic acid molecules derived from a cell-free biological sample of the subject.

Abstract: Methods for multiplex amplification of a plurality of targets of distinct sequence from a complex mixture are disclosed. In one aspect targets are circularized using a single circularization probe that is complementary to two regions in the target that flank a region to be amplified. The targets may hybridize to the circularization probe so that 5? or 3? flaps are generated and methods for removing flaps and circularizing the resulting product are disclosed. In another aspect targets are hybridized to dU probes so that 5? and 3? flaps are generated. The flaps are cleaved using 5? or 3? flap endonucleases or 3? to 5? exonucleases. The target sequences are then ligated to common primers, the dU probes digested and the ligated targets amplified.

Abstract: Provided herein are methods and systems for nucleic acid processing or analysis comprising generating a mixture comprising a first plurality of nucleic acid molecules derived from a biological sample of a subject, and a second plurality of nucleic acid molecules comprising sequences having at least one predetermined size; subjecting said first plurality of nucleic acid molecules, or derivative thereof; and second plurality of nucleic acid molecules or derivative thereof, to sequencing to generate a plurality of sequence reads; and processing said plurality of sequence reads to identify (i) a first set of sequence reads corresponding to at least a subset of said first plurality of nucleic acid molecules, and (ii) a second set of sequence reads corresponding to at least a subset of said second plurality of nucleic acid molecules, which second set of sequence reads corresponds to said sequences having said at least one predetermined size; and using said second set of sequence reads to identify one or more nuclei

Abstract: Provided herein are methods for processing a plurality of nucleic acid molecules derived from a cell-free biological sample, comprising bringing said plurality of nucleic acid molecules or derivatives thereof in contact with a plurality of binding agents, to provide a first subset of said plurality of nucleic acid molecules coupled to said plurality of binding agents and a second subset of said plurality of nucleic acid molecules; separating said first subset of said plurality of nucleic acid molecules coupled to said plurality of binding agents from said second subset of said plurality of nucleic acid molecules; circularizing a nucleic acid molecule derived from said first subset of said plurality of nucleic acid molecules to obtain a circularized nucleic acid molecule; and identifying said circularized nucleic acid molecule or derivative thereof.

Abstract: In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided. In some embodiments, methods comprise extending 3? ends of polynucleotides by adding one or more pre-determined nucleotides. In some embodiments, methods comprise use of a strand-tagging sequence.

Abstract: Provided herein are methods of cell-free nucleic acid size analysis. For example, methods herein can comprise preparing a first single stranded DNA library from a plurality of cell-free nucleic acid molecules from a subject and preparing a second single stranded DNA library from a plurality of cell-free nucleic acid molecules from a control.

Abstract: Provided herein are methods of determining that a subject has or is at risk of having a disease (e.g., cancer) using analysis of fragment enrichment or depletion on nucleic acid molecules derived from a cell-free biological sample of the subject.

Abstract: In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided. In some embodiments, methods comprise extending 3? ends of polynucleotides by adding one or more pre-determined nucleotides. In some embodiments, methods comprise use of a strand-tagging sequence.

Abstract: The invention is directed to methods for determining antigen-specific T cells. In some embodiments, methods of the inven-tion 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 off cells isolated based on their interaction with antigen, thereby form-ing 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 fre-quency increases in the latter sample relative to its frequency in the former sample.

Abstract: In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided.

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: Provided herein are methods of determining that a subject has or is at risk of having a disease (e.g., cancer) using nucleic acid molecules derived from a cell-free biological sample of the subject.

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: Provided herein are methods and systems for detecting non-metastatic cancer in a subject, comprising measuring a total cfDNA fragment size distribution for the plurality of cfDNA nucleic acid molecules of the subject and comparing the total cfDNA fragment size distribution for the plurality of cfDNA nucleic acid molecules of the subject to the total cfDNA fragment size distribution for the plurality of cfDNA nucleic acid molecules from a healthy control.

Abstract: In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided.

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: In some aspects, the present disclosure provides methods for identifying sequence variants in a nucleic acid sample. In some embodiments, a method comprises distinguishing between a true mutation in a polynucleotide and a random error introduced during an amplification step. In some embodiments, the methods reduce the number of false positives reported by a digital PCR assay. In some embodiments, the methods improve the accuracy of a digital PCR assay.

Abstract: In some aspects, the present disclosure provides methods for forming ligation products comprising single-stranded polynucleotides without the need for enriching the single-stranded polynucleotides. Ligation products formed by various aspects of the present disclosure can be useful for various applications, including but not limited to sequence analysis. In some embodiments, the ligation products comprise cell-free polynucleotides. In some aspects, the present disclosure provides reaction mixtures, kits and complexes consistent with the methods herein.

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: Methods for determining the methylation status of a plurality of cytosines are disclosed. In some aspects genomic DNA target sequences containing CpGs are targeted for analysis by multiplex amplification using target specific probes that can be specifically degraded prior to amplification. The targets may be modified with bisulfite prior to amplification. In another aspect targets are cut with methylation sensitive or insensitive restriction enzymes and marked with a tag using the target specific probes. The presence or absence of methylation may be determined using methylation sensitive restriction enzyme or bisulfite treatment. Detection in many embodiments employs hybridization to tag arrays, genotyping arrays or resequencing arrays.