Compositions and methods for immune repertoire monitoring
AssigneeLife Technologies Corporation
InventorsGeoffrey Lowman, Chenchen Yang, Timothy Looney
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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 B cell receptor heavy and light chains in a single assay, 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 amplification of rearranged genomic DNA (gDNA) sequences of a B cell receptor (BCR) repertoire in a sample, comprising:
- performing a single multiplex amplification reaction to amplify expressed target BCR nucleic acid template molecules using each of a set of:
- i) (a) a plurality of V gene primers directed to a majority of different V genes of BCR IgH coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene,
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the BCR IgH coding sequence;
- and
- ii) (a) a plurality of V gene primers directed to a majority of different V genes of BCR IgLlambda coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene,
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the BCR IgLlambda coding sequence;
- and
- iii) (a) a plurality of V gene primers directed to a majority of different V genes of BCR IgLkappa coding sequence comprising at least a portion of framework region 1 (FR3) within the V gene,
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the BCR IgLkappa coding sequence;
- and optionally
- iv) (a) one or more gene primers directed to a IgLkappa Cintron sequence, and
- (b) one or more gene primers directed to a KDE sequence;
- wherein each set of i) and ii) and iii) primers is directed to coding sequences of the same target BCR gene selected from an IgH, IgLlambda, and IgLkappa gene, respectively, and wherein performing the amplification using the set of i) and ii) and iii) primers results in amplicon molecules representing the target BCR repertoire in the sample;
- thereby generating target BCR amplicon molecules comprising the expressed target BCR repertoire.
A composition for analysis of a B cell receptor (BCR) 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 BCR IgH coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene,
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the BCR IgH coding sequence;
- and
- ii) (a) a plurality of V gene primers directed to a majority of different V genes of BCR IgLlambda coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene,
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the BCR IgLlambda coding sequence;
- and
- iii) (a) a plurality of V gene primers directed to a majority of different V genes of BCR IgLkappa coding sequence comprising at least a portion of framework region 1 (FR3) within the V gene,
- (b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the BCR IgLkappa coding sequence;
- and optionally
- iv) (a) one or more gene primers directed to a IgLkappa Cintron sequence, and
- (b) one or more gene primers directed to a KDE sequence;
- wherein each set of i) and ii) and iii) primers is directed to coding sequences of the same target BCR gene selected from an IgH, IgLlambda, and IgLkappa gene, respectively, and wherein performing the amplification using the set of i) and ii) and iii) primers results in amplicon molecules representing the target BCR repertoire in the sample.
Dependencies are inferred from the claim text.