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US 2025/0019864 A1Published application
Compositions and methods for immune repertoire sequencing
AssigneeLife Technologies Corporation
InventorsTimothy Looney, Geoffrey Lowman, Lifeng Lin
Life of this patent
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Abstract
The present disclosure provides methods, compositions, kits, and systems useful in the determination and evaluation of the immune repertoire. In one aspect, target-specific primer panels provide for the effective amplification of sequences of T cell receptor and/or B cell receptor chains with improved sequencing accuracy and resolution over the repertoire. Variable regions associated with the immune cell receptor are resolved to effectively portray clonal diversity of a biological sample and/or differences associated with the immune cell repertoire of a biological sample.
Claims20
A method for providing sequence of an immune receptor repertoire in a sample, comprising:
- I) performing sequencing of a target immune receptor repertoire library using a method for preparing the immune receptor repertoire library, comprising:
- A) generating target immune receptor amplicon molecules comprising performing a single multiplex amplification reaction to amplify expressed target immune receptor nucleic acid template molecules using at least one set of:
- i) (a) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 1 (FR1) within the V gene,
- (b) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 2 (FR2) within the V gene, or
- (c) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene;
- and
- ii) (a) one or more C gene primers directed to at least a portion of a C gene of the at least one immune receptor coding sequence, or
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the at least one immune receptor coding sequence;
- wherein each set of i) and ii) primers is directed to coding sequences of the same target immune receptor gene selected from a T cell receptor gene or an antibody receptor gene and wherein performing the amplification using the at least one set of i) and ii) primers results in amplicon molecules representing a target immune receptor repertoire in a sample, thereby generating immune receptor amplicon molecules;
- B) treating the amplicon molecules to form blunt-ended amplicon molecules;
- and
- C) ligating at least one adapter to at least one of the treated amplicon molecules, thereby producing a library of adapter-ligated target immune receptor amplicon molecules comprising the target immune receptor repertoire;
- wherein at least one of the plurality of V gene primers, the plurality of J gene primers and/or the one of more C gene primers includes two or more modified cleavable nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence;
- II) determining the sequence of the immune receptor amplicon molecules, wherein determining the sequence includes obtaining initial sequence reads, aligning the initial sequence read to a reference sequence, identifying productive reads, and correcting one or more indel errors to generate rescued productive sequence reads;
- and
- III) reporting the sequences of the expressed immune receptor molecules, thereby providing sequence of the immune receptor repertoire in the sample.
13 more elements
A method for amplification of expression nucleic acid sequences of an immune receptor repertoire in a sample, comprising:
- performing a single multiplex amplification reaction to amplify expressed target immune receptor nucleic acid template molecules using at least one set of:
- i) (a) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 1 (FR1) within the V gene,
- (b) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 2 (FR2) within the V gene, or
- (c) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene;
- and
- ii) (a) one or more C gene primers directed to at least a portion of a C gene of the at least one immune receptor coding sequence, or
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the at least one immune receptor coding sequence;
- wherein each set of i) and ii) primers is directed to coding sequences of the same target immune receptor gene selected from a T cell receptor gene or an antibody receptor gene and wherein performing the amplification using the at least one set of i) and ii) primers results in amplicon molecules representing the target immune receptor repertoire in the sample;
- thereby generating immune receptor amplicon molecules comprising the target immune receptor repertoire;
- wherein at least one of the plurality of V gene primers, the plurality of J gene primers and the one or more C gene primers has the following criteria:
- (1) includes two or more modified nucleotides having a cleavable group within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence;
- (2) length is about 15 to about 40 bases in length;
- (3) Tm of from above 60° C. to about 70° C.;
- (4) has low cross-reactivity with non-target sequences present in the sample;
- (5) at least the first four nucleotides (going from 3′ to 5′ direction) are non-complementary to any sequence within any other primer present in the same reaction;
- and
- (6) are non-complementary to any consecutive stretch of at least 5 nucleotides within any other produced target amplicon.
14 more elements
A composition for analysis of an immune repertoire in a sample, comprising at least one set of:
- i) (a) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 1 (FR1) within the V gene, or
- (b) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene;
- and
- ii) (a) one or more C gene primers directed to at least a portion of a C gene of the at least one immune receptor coding sequence, or
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the at least one immune receptor coding sequence;
- wherein each set of i) and ii) primers directed to coding sequences of the same target immune receptor gene selected from a T cell receptor or an antibody receptor;
- and
- wherein each set of i) and ii) primers directed to the same target immune receptor is configured to amplify the target immune receptor repertoire;
- and
- wherein at least one of the plurality of V gene primers, the plurality of J gene primers and/or the one of more C gene primers includes one or more cleavable groups located (i) near or at the termini of the primer or (ii) near or about the center nucleotide of the primer.
7 more elements
Dependencies are inferred from the claim text.