Methods of monitoring conditions by sequence analysis
View Patent ↗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. A method for monitoring an autoimmune disease, an infections disease or a cancer of an individual comprising:
(a) obtaining a sample from the individual comprising 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 complementarily 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 comprising at least 1000 sequence reads of at least 30 bp; and
(e) monitoring the autoimmune disease, infectious disease or cancer by determining levels of one or more CDR3 sequences that correlate with the disease or cancer.
2. The method of claim 1 wherein CDR3 sequences of said full repertoire of 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.
3. The method of claim 2 wherein said one or more correlating CDR3 sequences are distinguished among said full repertoire of CDR3 sequences by V, D, and J segments used.
4. The method of claim 2 wherein said sequences of CDR3 are from said immunoglobulin genes and have a level of somatic mutations and wherein said one or more correlating CDR3 sequences are distinguished among said full repertoire of CDR3 sequences by the level of somatic mutations in each of said one or more correlating CDR3 sequences.
5. The method of claim 2 wherein said CDR3 sequences each have a level within said full repertoire of CDR3 sequences and wherein said one or more correlating CDR3 sequences are distinguished among said full repertoire of CDR3 sequences by the level of each of said one or more correlating CDR3 sequences.
6. The method of any one of claims 1 to 5 wherein said sample comprises at least 1000 B-cells or at least 1000 T-cells.
7. The method of any one of claims 1 to 5 wherein said sample is blood.
8. The method of any one of claims 1 to 5 wherein said full repertoire of CDR3 sequences is from T-cell receptor β genes.