Methods of monitoring conditions by sequence analysis

Patent metadata

Field	Value
Publication number	US-10266901-B2
Application number	US-201614987165-A
Country	US
Kind code	B2
Filing date	Jan 4, 2016
Priority date	Nov 7, 2008
Publication date	Apr 23, 2019
Grant date	Apr 23, 2019

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.

First claim

What is claimed is:

1. A method for determining a profile of recombined T cell receptor (TCR) and/or immunoglobulin (IG) DNA sequences in a sample comprising T cells and/or B cells comprising:
a) amplifying said recombined TCR and/or IG DNA sequences in the sample by polymerase chain reaction (PCR) using a set of amplification primers, wherein said amplification primers comprise a generic primer sequence;
b) spatially isolating individual molecules of amplified recombined TCR and/or IG DNA comprising generic primer sequences;
c) amplifying the spatially isolated individual molecules of amplified recombined TCR and/or IG DNA by PCR using primers specific for the generic primer sequences wherein said primers specific for the generic primer sequence comprise a sequencing adaptor sequence;
d) sequencing said recombined TCR and/or IG DNA sequences by synthesis using reversible terminated labeled nucleotides to provide at least 1000 sequence reads each having an error rate and each comprising at least 30 base pairs so that different clonotypes of said sample are determined from sequence reads with a confidence of at least 99.9 percent based on the error rate; and
e) determining the levels of different clonotypes in the sample to generate a profile of clonotypes of recombined DNA sequences.

2. The method of claim 1 wherein said recombined TCR and/or IG DNA sequences in step (a) are cDNA sequences.

3. The method of claim 2 wherein the amplification primers are V segment and C segment primers.

4. The method of claim 3 wherein the primers are selected from the group consisting of TCR V region primers, TCR C region primers, IGH V region primers, and IGH C region primers.

5. The method of claim 4 wherein the TCR V region primers comprise any of SEQ ID NO. 1-34, wherein the TCR C region primer comprises SEQ ID NO: 135, wherein the IGH V region primers comprise any of SEQ ID NO: 39-126, and wherein IGH C region primers comprise any of SEQ ID NO: 127-129.

6. The method of claim 1 wherein the generic primer sequence comprises SEQ ID NO: 135.

7. The method of claim 1 wherein the sample comprising T cells and/or B cells is a bodily fluid selected from amniotic fluid surrounding a fetus, aqueous humor, bile, blood, blood plasma, cerumen, Cowper's fluid, chyle, chime, female ejaculate, interstitial fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubrication, vomit, water, feces, internal body fluids, cerebrospinal fluid, synovial fluid, intracellular fluid, and vitreous humor.

8. The method of claim 1 wherein the sample is a biopsy.

Assignees

• Adaptive Biotechnologies Corp

Inventors

• Faham Malek
• Willis Thomas

Classifications

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/118

  Prognosis of disease development · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title