Methods of monitoring conditions by sequence analysis
First Claim
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1. A method for quantitatively measuring clonotypes correlating to a disease state of a subject wherein the disease is a cancer, wherein the method comprises:
- (a) identifying a sequence and level of the clonotype correlated with the disease in the subject, comprising(1) generating and comparing a first clonotype profile from a first sample taken from the subject and a second clonotype profile from a second sample taken from the subject, wherein the first sample and the second sample each comprises T-cells and/or B-cells from the subject and are taken at different times from the subject, wherein each clonotype profile is generated by a method comprising;
(i) isolating genomic DNA from the first and second samples;
(ii) amplifying from said isolated genomic DNA recombined DNA sequences of the T-cells and/or B-cells in a multiplex PCR, comprising hybridizing to the genomic DNA a plurality of primers;
wherein the regions to be amplified comprise V, D, and/or J segments of an immunoglobulin and/or a T-cell or B-cell receptor gene;
(iii) spatially isolating individual molecules of the amplified sequences on a solid substrate, wherein the solid substrate is a glass slide; and
(iv) sequencing said amplified individual molecules to produce the clonotype profile by sequencing by synthesis using reversibly terminated labeled nucleotides;
(2) comparing the generated first and second clonotype profiles to identify the presence and level of the clonotype correlated with the disease in the subject; and
(b) producing a quantitative profile of clonotyes identified in step (a)(2) that correlate with the disease in the subject, wherein the profile comprises sequences and levels of said clonotypes.
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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.
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16 Claims
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1. A method for quantitatively measuring clonotypes correlating to a disease state of a subject wherein the disease is a cancer, wherein the method comprises:
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(a) identifying a sequence and level of the clonotype correlated with the disease in the subject, comprising (1) generating and comparing a first clonotype profile from a first sample taken from the subject and a second clonotype profile from a second sample taken from the subject, wherein the first sample and the second sample each comprises T-cells and/or B-cells from the subject and are taken at different times from the subject, wherein each clonotype profile is generated by a method comprising; (i) isolating genomic DNA from the first and second samples; (ii) amplifying from said isolated genomic DNA recombined DNA sequences of the T-cells and/or B-cells in a multiplex PCR, comprising hybridizing to the genomic DNA a plurality of primers;
wherein the regions to be amplified comprise V, D, and/or J segments of an immunoglobulin and/or a T-cell or B-cell receptor gene;(iii) spatially isolating individual molecules of the amplified sequences on a solid substrate, wherein the solid substrate is a glass slide; and (iv) sequencing said amplified individual molecules to produce the clonotype profile by sequencing by synthesis using reversibly terminated labeled nucleotides; (2) comparing the generated first and second clonotype profiles to identify the presence and level of the clonotype correlated with the disease in the subject; and (b) producing a quantitative profile of clonotyes identified in step (a)(2) that correlate with the disease in the subject, wherein the profile comprises sequences and levels of said clonotypes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification