Methods of monitoring conditions by sequence analysis

• US 9,228,232 B2
• Filed: 07/11/2014
• Issued: 01/05/2016
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Sequenta LLC
• Inventors: Faham, Malek, Willis, Thomas
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US14/329,873
• Publication Number:
  US 20140342367A1
• Time in Patent Office: 543 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

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

1. A method for monitoring an autoimmune disease, an infectious disease or a cancer of an individual by correlating clonotypes unique to the individual, the method comprising:

(a) amplifying from a sample of nucleic acids from T cells and/or B cells and/or cell free DNA or RNA from the individual in a multiplex polymerase chain reaction (PCR) recombined nucleic acids comprising complementary determining region 3 (CDR3) sequences from T cell receptor genes or immunoglobulin genes;

(b) spatially isolating individual molecules of the amplified recombined nucleic acids on a solid surface;

(c) sequencing by synthesis using reversibly terminated labeled nucleotides die spatially isolated recombined nucleic acids to generate at least 10,000 sequence reads each having an error rate and at least 30 bp;

(d) combining the sequence reads into clonotypes of the recombined nucleic acids, wherein sequence reads are combined into different clonotypes whenever said sequence reads are distinct with a confidence of at least 99.9 percent based on error rates, frequencies and base divergences of the sequence reads;

(e) determining levels of clonotypes by counting sequence reads thereof; and

(f) monitoring the autoimmune disease, infectious disease or cancer from the levels of one or more correlating clonotypes of the autoimmune disease, infectious disease or cancer unique to the individual.

2. The method of claim 1 wherein said step of sequencing produces paired end reads and wherein said clonotypes are combined from the paired end reads.

3. The method of claim 1 wherein said step of sequencing includes re-amplifying said spatially isolated molecules of recombined nucleic acid.

4. The method of claim 1 wherein said step of sequencing comprises providing at least 100,000 sequence reads per run.

5. The method of claim 1 wherein said step of sequencing includes creating clusters of said spatially isolated recombined nucleic acids on said solid surface.

6. The method of claim 1 wherein said step of combining includes estimating a likelihood that a base of a sequence read is a sequencing error based on a quality score of the base.

7. The method of claim 1 wherein said sample comprises at least 10⁵ B-cells or at least 10⁵ T-cells and wherein said step of sequencing comprises at least 10⁶ reads per run.

8. A method for monitoring an autoimmune disease, an infectious disease or a cancer of an individual by correlating clonotypes unique to the individual, the method comprising:

(a) amplifying from a sample comprising T-cells and/or B-cells and/or cell-free DNA or RNA from the individual in a multiplex polymerase chain reaction (PCR) molecules of recombined nucleic acid comprising complementary determining region 3 (CDR3) sequences from T-cell receptor genes or immunoglobulin genes;

(b) spatially isolating individual molecules of the amplified recombined nucleic acids;

(c) sequencing by synthesis the spatially isolated recombined nucleic acids to provide sequence reads of CDR3 sequences, wherein each sequence read has an error rate and wherein said sequencing includes incorporating by a polymerase one or more nucleoside triphosphates at the end of a sequencing primer hybridized to said recombined nucleic acids and detection thereof by a change in current;

(d) combining the sequence reads into clonotypes of the recombined nucleic acids, wherein sequence reads are coalesced into different clonotypes whenever said sequence reads are distinct with a confidence of at least 99.9 percent based on error rates, frequencies and base divergences of the sequence reads;

(e) determining levels of clonotypes by counting sequence reads thereof; and

(f) monitoring said disease from the levels of one or more correlating clonotypes unique to the individual among the CDR3 sequences in the sample.

9. The method of claim 8 wherein said sample is blood.

10. The method of claim 8 wherein said step of sequencing provides at least 10,000 sequence reads and wherein said change in current is detected by a chemical-sensitive field effect transistor.

11. The method of claim 8 wherein said step of combining includes estimating a likelihood that a base of a sequence read is a sequencing error based on a quality score of the base.

12. The method of claim 8 wherein said sample comprises at least 10⁵ B-cells or at least 10⁵ T-cells and wherein said step of sequencing comprises at least 10⁶ sequence reads per run.

13. The method of claim 8 wherein said step of sequencing comprises generating at least 10,000 sequence reads per run and wherein said step of spatially isolating includes attaching said individual molecules of recombined nucleic acid to beads and transferring the beads to individual reaction chambers on a chemFET array.

14. The method of claim 8 wherein said step of spatially isolating includes dispersing said individual molecules into compartments of a water/oil emulsion to obtain one molecule in each compartment.

15. A method for monitoring an autoimmune disease, an infectious disease or a cancer of an individual by one or more correlating clonotypes unique to the individual, the method comprising:

(a) amplifying from a sample of nucleic acids from B-cells and/or cell free DNA or RNA from the individual in a multiplex polymerase chain reaction (PCR) recombined nucleic acids comprising complementary determining region 3 (CDR3) sequences from immunoglobulin genes, wherein each CDR3 sequence comprises a V segment;

(b) spatially isolating individual molecules of the amplified recombined nucleic acids on a solid surface;

(c) sequencing by synthesis using reversibly terminated labeled nucleotides the spatially isolated recombined nucleic acids to generate at least 10,000 sequence reads each having at least 30 bp and each having an error rate;

(d) combining the sequence reads of the recombined nucleic acids to identify clonotypes with V segments and somatic hypermutations therein with a confidence of at least 99.9 percent based on error rates, frequencies and base divergences of the sequence reads;

(e) determining levels of clonotypes by counting sequence reads thereof; and

(f) monitoring the autoimmune disease, infectious disease or cancer from the levels of one or more correlating clonotypes of the autoimmune disease, infectious disease or cancer unique to the individual.

16. The method of claim 15 wherein said CDR3 sequences are from said immunoglobulin genes and have a level of somatic mutations and wherein said correlating clonotypes of said autoimmune disease, infectious disease or cancer are distinguished among said CDR3 sequences by the level of somatic mutations in their clonotypes.

17. The method of claim 15 wherein said sequence reads are combined into different clonotypes whenever said sequence reads are distinct with a confidence of at least 99.9 percent.

18. The method of claim 15 wherein said step of sequencing produces paired end reads and wherein said clonotypes are combined from the paired end reads.

19. The method of claim 15 wherein said step of sequencing includes re-amplifying said spatially isolated molecules of recombined nucleic acid.

20. The method of claim 15 wherein said step of sequencing includes creating clusters of said spatially isolated recombined nucleic acids on said solid surface.

21. The method of claim 15 wherein said step of combining includes estimating a likelihood that a base of a sequence read is a sequencing error based on a quality score of the base.

22. The method of claim 15 wherein said step of monitoring includes determining said clonotypes correlated with said autoimmune disease, infectious disease or cancer with at least a frequency of 0.1 percent.

23. The method of claim 15 wherein said step of amplifying includes a multiplex PCR wherein each of said V segments has multiple primers.