Method for genotyping clonotype profiles using sequence tags

• US 10,526,650 B2
• Filed: 08/02/2018
• Issued: 01/07/2020
• Est. Priority Date: 07/01/2013
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Adaptive Biotechnologies Corp.
• Inventors: Asbury, Thomas, Hervold, Kieran, Kotwaliwale, Chitra, Faham, Malek, Moorhead, Martin, Weng, Li, Wittkop, Tobias, Zheng, Jianbiao
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US16/053,118
• Publication Number:
  US 20190040462A1
• Time in Patent Office: 523 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16H 50/30 for calculating health indices; for individual health risk assessment
• CPC Class Codes: G16Z 99/00 Subject matter not provided for in other main groups of this subclass
• 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

The invention is directed to sequence-based profiling of populations of nucleic acids by multiplex amplification and attachment of one or more sequence tags to target nucleic acids and/or copies thereof followed by high-throughput sequencing of the amplification product. In some embodiments, the invention includes successive steps of primer extension, removal of unextended primers and addition of new primers either for amplification (for example by PCR) or for additional primer extensions. Some embodiments of the invention are directed to minimal residual disease (MRD) analysis of patients being treated for cancer. Sequence tags incorporated into sequence reads provide an efficient means for determining clonotypes and at the same time provide a convenient means for detecting carry-over contamination from other samples of the same patient or from samples of a different patient which were tested in the same laboratory.

Claims

1. A method for detecting and measuring a presence, absence and/or level of sequence tags from one or more previous samples in a sample, comprising:

(a) attaching sequence tags to cancer genes in a sample obtained from an individual to form tag-nucleic acid conjugates, wherein at least one of the cancer genes or copies thereof has a different sequence tag attached, and wherein the cancer genes from the sample are characteristic of a cancer of the individual, wherein the attaching comprises;

(i) combining in a reaction mixture under primer extension conditions a first set of primers with the sample, wherein each primer of the first set comprises sequence complementary to a cancer gene, a 5′

-non-complementary end containing a first primer binding site and a sequence tag disposed between the sequence complementary to the cancer gene and the first primer binding site, wherein the sequence complementary to a cancer gene of each primer from the first set anneals to a different cancer gene at a first predetermined location and is extended to form a first extension product; and

(ii) adding to the reaction mixture under primer extension conditions a second set of primers, wherein each primer of the second set has sequence complementary to a cancer gene, wherein the sequence complementary to the cancer gene anneals to the first extension product at a second predetermined location, and wherein each primer of the second set is extended to form a second extension product, wherein each second extension product comprises a first primer binding site, sequence tag, and cancer gene sequence;

(b) amplifying the tag-nucleic acid conjugates;

(c) sequencing the amplified tag-nucleic acid conjugates to generate sequence reads for each of the amplified tag-nucleic acid conjugates, wherein each of the sequence reads has an error rate, and wherein each of the sequence reads comprises a tag sequence and a cancer gene sequence;

(d) comparing the sequence reads for each of the amplified tag-nucleic acid conjugates to separately determined tag sequences from other samples; and

(e) determining the presence, absence and/or level of sequence tags from one or more previous samples by the identity of one or more tag sequences with any separately determined tag sequences from the one or more previous samples.

2. The method of claim 1, further comprising aligning the sequence reads having like sequence tags to form groups of sequence reads having the same sequence tags;

and coalescing cancer gene sequence reads of groups to determine sequences of cancer genes, wherein groups of sequence reads are coalesced into different cancer gene molecules whenever said groups of cancer gene sequences are distinct with a likelihood of at least ninety-five percent.

3. The method of claim 1, wherein the amplifying comprises performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain reaction using forward primers specific for the first primer binding site and reverse primers specific for the second set of primers.

4. The method of claim 1, further comprising a step of removing non-extended primers of the first set and/or the second set after the first extension product and/or after the second extension product is formed.

5. The method of claim 1, wherein the annealing and extension of the primers of the first set is repeated after melting the first extension product.

6. The method of claim 1, wherein the annealing and extension of the primers of the second set is repeated after melting the second extension product.

7. The method of claim 1, wherein the individual is suffering from a lymphoid proliferative disorder.

8. The method of claim 1, wherein the individual is suffering from a non-lymphoid or non-myeloid cancer.

9. The method of claim 1, wherein the cancer genes are one or more of the cancer genes found in Table 1.

10. The method of claim 1, wherein the other samples are samples obtained from the same individual.

11. The method of claim 1, wherein the other samples are samples obtained from a different individual.

21. The method of claim 7, wherein the lymphoid proliferative disorder is lymphoid cancer.

12. A method for detecting and measuring a presence, absence and/or level of sequence tags from one or more previous samples in a sample, comprising:

(a) attaching sequence tags to recombined nucleic acids from B-cells and/or T-cells in a sample obtained from an individual to form tag-nucleic acid conjugates, wherein at least one of the recombined nucleic acids from B-cells and/or T-cells or copies thereof has a different sequence tag attached, wherein the attaching comprises;

(i) combining in a reaction mixture under primer extension conditions a first set of primers with a sample of recombined nucleic acids from B-cells and/or T-cells and/or cell-free DNA, wherein each primer of the first set comprises a receptor-specific portion, a 5′

-non-complementary end containing a first primer binding site and a sequence tag disposed between the receptor-specific portion and the first primer binding site, wherein the receptor-specific portion anneals to a different recombined nucleic acid at a first predetermined location and is extended to form a first extension product; and

(ii) adding to the reaction mixture under primer extension conditions a second set of primers, wherein each primer of the second set has a receptor-specific portion, wherein the receptor-specific portion anneals to the first extension product at a second predetermined location, and wherein each primer of the second set is extended to form a second extension product, wherein each second extension product comprises a first primer binding site, a sequence tag, and recombined nucleic acid encoding a portion of a T cell receptor chain or a B cell receptor chain;

(b) amplifying the tag-nucleic acid conjugates;

(c) sequencing a sample of the tag-nucleic acid conjugates to provide sequence reads each comprising a tag sequence and a recombined nucleic acid sequence;

(d) comparing the sequence reads for each of the amplified tag-nucleic acid conjugates to separately determined tag sequences from other samples; and

(e) determining the presence, absence and/or level of sequence tags from one or more previous samples by the identity of one or more tag sequences with any separately determined tag sequences from the one or more previous samples.

13. The method of claim 12, further comprising aligning sequence reads having like tag sequences to form groups of sequence reads having identical sequence tags;

and coalescing recombined nucleic acid sequences of groups to determine clonotypes, wherein groups of sequence reads are coalesced into different clonotypes whenever said groups of recombined nucleic acid sequences are distinct with a likelihood of at least 99.9 percent.

14. The method of claim 12, wherein the amplifying comprises performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain reaction using forward primers specific for the first primer binding site and reverse primers specific for the second set of primers.

15. The method of claim 12, further comprising a step of removing non-extended primers of the first set and/or the second set after the first extension product and/or after the second extension product is formed.

16. The method of claim 12, wherein the annealing and extension of the primers of the first set is repeated after melting the first extension product.

17. The method of claim 12, wherein the annealing and extension of the primers of the second set is repeated after melting the second extension product.

18. The method of claim 12, wherein the other samples are samples obtained from the same individual.

19. The method of claim 12, wherein the other samples are samples obtained from a different individual.

20. A method of monitoring a minimal residual disease of a cancer comprising:

(a) attaching sequence tags to each of a plurality of cancer genes in a sample obtained from an individual to form tag-nucleic acid conjugates, wherein at least one of the cancer genes or copies thereof has a different sequence tag attached, and wherein the cancer genes from the sample are characteristic of a cancer of the individual, wherein the attaching comprises;

(i) combining in a reaction mixture under primer extension conditions a first set of primers with the sample, wherein each primer of the first set comprises sequence complementary to a cancer gene, a 5′

-non-complementary end containing a first primer binding site and a sequence tag disposed between the sequence complementary to the cancer gene and the first primer binding site, wherein the sequence complementary to a cancer gene of each primer from the first set anneals to a different cancer gene at a first predetermined location and is extended to form a first extension product; and

(ii) adding to the reaction mixture under primer extension conditions a second set of primers, wherein each primer of the second set has sequence complementary to a cancer gene, wherein the sequence complementary to the cancer gene anneals to the first extension product at a second predetermined location, and wherein each primer of the second set is extended to form a second extension product, wherein each second extension product comprises a first primer binding site, sequence tag, and cancer gene sequence;

(b) amplifying the tag-nucleic acid conjugates;

(c) sequencing the amplified tag-nucleic acid conjugates to generate sequence reads for each of the amplified tag-nucleic acid conjugates, wherein each of the sequence reads has an error rate, and wherein each of the sequence reads comprises a tag sequence and a cancer gene sequence;

(d) aligning sequence reads having like tag sequences to form groups of sequence reads having the same sequence tags;

(e) coalescing cancer gene sequences of groups to determine sequences of cancer gene molecules, wherein groups of sequence reads are coalesced into different cancer gene molecules whenever said groups of cancer gene sequences are distinct with a likelihood of at least ninety-five percent; and

(f) detecting in a profile of the cancer gene molecules the presence, absence and/or level of cancer gene molecules characteristic of the cancer of the individual.