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 determining a profile of clonotypes of recombined DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) spatially isolating individual molecules of recombined DNA sequences from said cells on a solid surface;
(c) sequencing said spatially isolated individual molecules of recombined DNA sequences to provide at least 1000 sequence reads each comprising at least 30 bp so that different clonotypes of said sample are determined from such sequence reads with a confidence of at least 99.9 percent; and
(d) determining levels of different clonotypes from said sample to generate said profile of clonotypes of recombined DNA sequences.
2. A method for determining a profile of clonotypes of recombined DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) spatially isolating individual molecules of recombined DNA sequences from said cells on a solid surface;
(c) sequencing said spatially isolated individual molecules of recombined DNA sequences to provide at least 1000 sequence reads each comprising at least 30 bp, wherein different clonotypes of said sample are determined from such sequence reads with a confidence of at least 99.9 percent and wherein said sequencing does not comprise pyrosequencing; and
(d) determining levels of different clonotypes from said sample to generate said profile of clonotypes of recombined DNA sequences.
3. A method for determining a profile of clonotypes of recombined T-cell receptor gene and/or immunoglobulin gene DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) spatially isolating individual molecules of genomic DNA from said cells on a solid surface;
(c) sequencing said spatially isolated individual molecules of genomic DNA to provide at least 1000 sequence reads each comprising at least 30 bp so that different clonotypes of said sample are determined from such sequence reads with a confidence of at least 99.9 percent; and
(d) determining levels of different clonotypes from said sample to generate said profile of clonotypes of recombined T-cell receptor gene and/or immunoglobulin gene DNA sequences.
4. The method of any one of claims 1 to 3 , wherein the individual molecules comprise a repertoire off-cell receptor genes and/or B-cell receptor genes.
5. The method of any one of claims 1 to 4 , wherein the solid surface is provided by a glass slide or beads.
6. The method of claim 1 or claim 2 , wherein the molecules of recombined DNA sequences are amplified.
7. The method of claim 3 , wherein the molecules of genomic DNA are amplified.
8. The method of any of claims 1 to 3 , wherein said sequencing comprises sequencing by synthesis using reversibly terminated labeled nucleotides.
9. The method of any of claims 1 to 3 , wherein said sample is taken from a subject having or suspected of having systemic lupus erythematosus.
10. The method of any one of claims 1 to 3 , further comprising determining one or more correlating clonotypes in the subject based on the profile of recombined DNA sequences in T-cells and/or B-cells.
11. The method of claim 10 wherein said determining one or more correlating clonotypes comprises comparing clonotype profiles from at least two samples.
12. The method of claim 10 , wherein said sample is from a tissue affected by a disease.
13. The method of claim 10 , wherein said sample is from one or more individuals at a peak state of a disease.
14. The method of claim 13 , wherein said one or more correlating clonotypes are present in the peak state of the disease.
15. The method of claim 10 , wherein said T-cells and/or B-cells comprise a subset of T-cells and/or B-cells.
16. The method of claim 15 , wherein said subset of T-cells and/or B-cells are enriched by interaction with a marker.
17. The method of claim 16 , wherein said marker is a cell surface marker on the subset of T-cells and/or B-cells.
18. The method of claim 17 , wherein said subset of T-cells and/or B-cells interact with an antigen specifically present in a disease.
19. The method of any one of claims 1 to 3 wherein said step of sequencing includes providing a number of said sequence reads which depends on base quality scores of said sequence reads.
20. The method of any one of claims 1 to 3 wherein said step of sequencing includes providing at least 10,000 said sequence reads each comprising at least 30 bp.