# US 10,246,752 B2 · Methods of monitoring conditions by sequence analysis

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- Number: US 10,246,752 B2
- Document ID: US10246752B2
- Status: Granted
- Kind: B2
- Application number: 15/820,655
- Filed: 2017-11-22
- Published: 2018-04-26
- Granted: 2019-04-02
- Earliest claimed: 2008-11-07
- Assignees: Adaptive Biotechnologies Corporation
- Applicants: Adaptive Biotechnologies Corp.
- Inventors: Malek Faham; Thomas Willis
- Web page: https://patentgrep.com/patent/US10246752B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- Y02A 90/10: Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein each of the clonotype profiles comprises at least 1000 sequence reads, wherein each read is about 30 base pairs to about 300 base pairs, so that different clonotypes of said sample are determined from such sequence reads with a confidence of at least 99.9 percent.

### Claim 3 (depends on 1)

The method of claim 1, wherein the step (a)(1) further comprises minimizing errors introduced by amplification and/or by sequencing, comprising combining sequence reads together to more accurately determine the frequency of individual molecules.

### Claim 4 (depends on 1)

The method of claim 1, wherein the region to be amplified comprises V, D, and/or J segments of an immunoglobulin gene.

### Claim 5 (depends on 1)

The method of claim 1, wherein the region to be amplified comprises V, D, and/or J segments of a T cell receptor gene.

### Claim 6 (depends on 4)

The method of claim 4, wherein the immunoglobulin gene is an IgH gene.

### Claim 7 (depends on 6)

The method of claim 6, wherein the primers bind to a V segment and a J segment of the IgH gene.

### Claim 8 (depends on 4)

The method of claim 4, wherein the immunoglobulin gene is an IgK gene.

### Claim 9 (depends on 8)

The method of claim 8, wherein the primers bind to a V segment and a J segment of the IgK gene.

### Claim 10 (depends on 4)

The method of claim 4, wherein the immunoglobulin gene is an IgL gene.

### Claim 11 (depends on 10)

The method of claim 10, wherein the primers bind to a V segment and a J segment of the IgL gene.

### Claim 12 (depends on 5)

The method of claim 5, wherein the T cell receptor gene is a T-cell receptor gamma (TRG) gene.

### Claim 13 (depends on 12)

The method of claim 12, wherein the primers bind to a V segment and a J segment of a T-cell receptor gamma (TRG) gene.

### Claim 14 (depends on 5)

The method of claim 5, wherein the T-cell receptor gene is a T-cell receptor beta (TRB) gene.

### Claim 15 (depends on 14)

The method of claim 14, wherein the primers bind to a V segment and a J segment of a T-cell receptor beta (TRB) gene.

### Claim 16 (depends on 1)

The method of claim 1, wherein step b) further comprises determining the state of disease in the subject, comprising predicting the disease state using the set of factors which include one or more of the number of the number of positive clonotypes, the level of a positive clonotype, the rate of change of a clonotype, the level of a clonotype at episode peak, and the level of a clonotype at an inactive disease state.