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. A method for determining one or more correlating clonotypes of a disease unique to a subject comprising:
(a) generating one or more clonotype profiles from at least two samples, wherein at least one sample is related to a first state of the disease, and
(b) determining one or more correlating clonotypes unique to the subject based on comparing a clonotype profile of the at least one sample with one or more clonotype profiles of at least one other sample;
wherein each of said clonotype profiles is generated by the following steps:
(ii) spatially isolating individual molecules of recombined DNA sequences from T cells and/or B cells from the subject on a solid substrate;
(iii) amplifying molecules of the recombined DNA sequences on the solid substrate to form clusters;
(iv) sequencing by synthesis using reversibly terminated labeled nucleotides said recombined DNA sequences of the clusters to provide at least 1000 sequence reads each having an error rate;
(v) coalescing sequence reads of different clusters into different clonotypes of the recombined DNA sequences of the sample whenever the sequence reads are distinct with a confidence of at least 99.9 percent based on the error rate; and
(vi) determining a level of each of the different clonotypes of the recombined DNA sequences from said sample to generate said clonotype profile.
2. The method of claim 1 , wherein said at least one sample is from a tissue affected by the disease.
3. The method of claim 1 or claim 2 , wherein the disease is systemic lupus erythematosus or multiple sclerosis.
4. The method of claim 1 wherein said sample comprises at least 10,000 B-cells.
5. The method of claim 4 wherein said sample comprises at least 100,000 B-cells.
6. The method of claim 1 wherein said sample comprises at least 10,000 T-cells.
7. The method of claim 1 wherein said step of sequencing provides at least 100,000 sequence reads.
8. The method of claim 7 wherein said step of sequencing provides at least 1,000,000 sequence reads.
9. The method of any of claims 1 to 8 wherein said recombined DNA sequences comprise complementary determining region 3 (CDR3).
10. The method of any of claims 1 to 9 wherein said sequence reads each comprise at least 30 bp.
11. The method of claim 10 wherein said sample is a blood sample.
12. The method of claim 1 wherein said step of coalescing includes determining distinct sequence reads by their frequencies and base divergence.
13. The method of claim 1 wherein said step of coalescing includes estimating a likelihood that a base of a sequence read is a sequencing error based on a quality score of the base.