Method for genotyping clonotype profiles using sequence tags
First Claim
1. A method of detecting minimal residual disease in a patient being treated for cancer, the method comprising the steps of:
- (a) attaching sequence tags to each of a plurality of recombined nucleic acids in a sample containing T-cells and/or B-cells and/or cell free DNA or RNA obtained from the patient to form tag-nucleic acid conjugates, wherein at least one recombined nucleic acid or copies thereof have different sequence tags attached and are characteristic of the cancer of the patient, and 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 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, 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) aligning sequence reads having like tag sequences to form groups of sequence reads having identical sequence tags;
(e) 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; and
(f) detecting in a clonotype profile the presence, absence and/or level of clonotypes correlated with the cancer of the patient, thereby detecting the minimal residual disease in a patient.
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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.
391 Citations
16 Claims
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1. A method of detecting minimal residual disease in a patient being treated for cancer, the method comprising the steps of:
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(a) attaching sequence tags to each of a plurality of recombined nucleic acids in a sample containing T-cells and/or B-cells and/or cell free DNA or RNA obtained from the patient to form tag-nucleic acid conjugates, wherein at least one recombined nucleic acid or copies thereof have different sequence tags attached and are characteristic of the cancer of the patient, and 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 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, 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) aligning sequence reads having like tag sequences to form groups of sequence reads having identical sequence tags; (e) 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; and (f) detecting in a clonotype profile the presence, absence and/or level of clonotypes correlated with the cancer of the patient, thereby detecting the minimal residual disease in a patient. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification