METHODS OF MONITORING CONDITIONS BY SEQUENCE ANALYSIS
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.
1 - 34 . (canceled)
35 . The method of claim 65 , wherein said disease is an autoimmune disease and said one or more correlating clonotypes are present in a peak state of the disease, wherein the peak state of the disease is a flare state of the autoimmune disease.
36 - 39 . (canceled)
40 . The method of claim 65 , wherein said T-cells and/or B-cells comprise a subset of T-cells and/or B cells.
41 . The method of claim 40 , wherein said subset of T-cells and/or B-cells are enriched by interaction with a marker.
42 . The method of claim 41 , wherein said marker is a cell surface marker on the subset of T-cells and/or B-cells.
43 . The method of claim 40 , wherein said subset of T-cells and/or B-cells interact with an antigen specifically present in the disease.
44 . The method of claim 35 , wherein the disease is systemic lupus erythematosus or multiple sclerosis.
45 - 64 . (canceled)
65 . A method for monitoring an autoimmune disease, an infectious disease or a cancer of an individual, the method comprising:
(a) obtaining a sample from the individual comprising T-cells and/or B-cells;
(b) spatially isolating individual molecules of nucleic acids from said cells of the sample, the individual molecules of nucleic acid comprising sequences of complementary determining region 3 (CDR3) from T-cell receptor genes or immunoglobulin genes;
(c) sequencing the spatially isolated individual molecules of the nucleic acids to provide a full repertoire of CDR3 sequences; and
(d) monitoring the autoimmune disease, infectious disease or cancer by determining levels of correlating clonotypes in the full repertoire of CDR3 sequences from the sample.
66 . The method of claim 65 wherein each of said full repertoire of CDR3 sequences comprises at least 1000 sequence reads each comprising at least 30 bp.
67 . The method of claim 66 further including a step of amplifying said individual molecules of nucleic acid prior to said step of spatially isolating said individual molecules.
68 . The method of claim 67 wherein said CDR3 sequences each include a V segment and wherein said step of amplifying includes amplifying in a polymerase chain reaction using primers specific for each of the V segments.
69 . The method of claim 66 wherein said one or more correlating clonotypes are distinguished among said full repertoire of CDR3 sequences by V, D, and J segments used.
70 . The method of claim 66 wherein said sequences of CDR3 are from said immunoglobulin genes and have a level of somatic mutations and wherein said one or more correlating clonotypes are distinguished among said full repertoire of CDR3 sequences by the level of somatic mutations in each of such clonotypes.
71 . The method of claim 66 wherein said sequences of CDR3 each have a level within said full repertoire of CDR3 sequences and wherein said one or more correlating clonotypes are distinguished among said full repertoire of CDR3 sequences by the level of each of such clonotypes.
72 . The method of claim 66 wherein said monitoring is for a recurrence of a cancer and wherein said correlating clonotypes are from an immune response to the cancer.
73 . The method of claim 65 wherein each of said samples comprises at least 10,000 B-cells and/or T-cells.
74 . The method of claim 72 wherein said sample is blood.
75 . The method of claim 65 wherein said sample comprises cell-free DNA or RNA.
76 . The method of claim 65 wherein said sample comprises at least 10 5 B-cells or at least 10 5 T-cells and wherein said step of sequencing comprises at least 10 6 reads per run.
77 . A method for monitoring an autoimmune disease, an infectious disease or a cancer of an individual, the method comprising:
(a) obtaining a sample of nucleic acids from T-cells and/or B-cells of an individual;
(b) spatially isolating individual molecules of nucleic acids from said cells of the sample, the individual molecules of nucleic acid comprising sequences of complementary determining region 3 (CDR3) from T-cell receptor genes or immunoglobulin genes;
(c) sequencing the spatially isolated individual molecules of the nucleic acids to generate sequence reads of CDR3 sequences;
(d) coalescing the sequence reads into clonotypes to form a clonotype profile; and
(c) monitoring the autoimmune disease, infectious disease or cancer by determining levels of correlating clonotypes in the clonotype profile.
78 . The method of claim 77 wherein said step of sequencing comprises sequencing by synthesis said spatially isolated individual molecules.
79 . The method of claim 78 wherein said sequencing by synthesis includes using reversibly terminated labeled nucleotides.
80 . The method of claim 78 wherein said step of sequencing comprises at least 1000 reads per run.
81 . The method of claim 77 further including a step of amplifying said individual molecules of nucleic acid prior to said step of spatially isolating said individual molecules.
82 . The method of claim 81 wherein said step of sequencing comprises at least 1000 reads per run.
83 . The method of claim 77 wherein said step of spatially isolating individual molecules includes spatially isolating said individual molecules of said nucleic acids in two dimensions on a solid substrate.
84 . The method of claim 77 wherein said sample comprises at least 10 5 B-cells or at least 10 5 T-cells and wherein said step of sequencing comprises at least 10 6 reads per run.
85 . A method for monitoring an autoimmune disease, an infectious disease or a cancer of an individual by determining levels of correlating clonotypes of the disease, the method comprising:
(a) obtaining a sample from the individual comprising at least 10,000 T-cells and/or B-cells;
(b) amplifying molecules of nucleic acid from said cells of the sample, the molecules of nucleic acid comprising sequences of complementary determining region 3 (CDR3) from T-cell receptor genes or immunoglobulin genes;
(c) spatially isolating individual molecules of the amplified nucleic acids;
(d) sequencing the spatially isolated individual molecules of the amplified nucleic acids to provide a full repertoire of CDR3 sequences; and
(e) monitoring said disease by determining levels of the one or more correlating clonotypes among the full repertoire of CDR3 sequences in the sample.
86 . The method of claim 85 wherein said sample is blood.
87 . The method of claim 85 wherein each of said full repertoire of CDR3 sequences comprises at least 1000 sequence reads each comprising at least 30 bp.
88 . The method of claim 85 wherein said full repertoire of CDR3 sequences comprise a full repertoire of CDR3 sequences of T-cell receptor β genes of said individual.
89 . The method of claim 85 wherein said sample comprises at least 10 5 B-cells or at least 10 5 T-cells and wherein said step of sequencing comprises at least 10 6 reads per run.
90 . The method of claim 85 wherein said step of sequencing comprises generating at least 1000 reads per run and coalescing reads to form clonotypes.
91 . The method of claim 85 wherein said step of sequencing comprises sequencing by synthesis said spatially isolated individual molecules.
92 . The method of claim 35 further including said step of treating said individual with a drug based on said levels of said one or more correlating clonotypes.
93 . The method of claim 35 wherein said autoimmune disease is rheumatoid arthritis and wherein said step of treating includes treating said individual with anti-inflammatory drugs or DMARDs.
94 . The method of claim 35 wherein said autoimmune disease is ankylosing spondylitis and wherein said step of treating includes treating said individual with a drug selected from the group consisting of anti-inflammatory drugs, DMARDs, or TNFα blockers.