Identification of antigen-specific B cell receptors
First Claim
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1. A method for identifying antigen-specific B-cell receptor (BCR) sequences comprising:
- (A) incubating a plurality of B-cells with an antigen library displayed by an organism capable of displaying antigens;
(B) distributing the B-cells bound to antigens of the antigen library into a plurality of aliquots;
(C) isolating nucleic acids from B-cells bound to antigens of the antigen library and from the organism displaying said antigens;
(D) sequencing the following elements from each of the aliquots;
(i) B-cell heavy chain sequence,(ii) B-cell light chain sequence, and(iii) a nucleotide sequence encoding the antigen bound to the BCR; and
(E) identifying the sequenced elements of (D) that occur together in more than one aliquot thereby identifying antigen-specific BCR sequences.
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Abstract
Compositions and methods are disclosed for identifying B-cell receptor sequences that bind to corresponding antigens. The disclosed methods and related embodiments permit the identification paired relationships between rearranged gene segments of B-cell receptors with unique antigens.
363 Citations
30 Claims
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1. A method for identifying antigen-specific B-cell receptor (BCR) sequences comprising:
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(A) incubating a plurality of B-cells with an antigen library displayed by an organism capable of displaying antigens; (B) distributing the B-cells bound to antigens of the antigen library into a plurality of aliquots; (C) isolating nucleic acids from B-cells bound to antigens of the antigen library and from the organism displaying said antigens; (D) sequencing the following elements from each of the aliquots; (i) B-cell heavy chain sequence, (ii) B-cell light chain sequence, and (iii) a nucleotide sequence encoding the antigen bound to the BCR; and (E) identifying the sequenced elements of (D) that occur together in more than one aliquot thereby identifying antigen-specific BCR sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. A method for identifying antigen-specific B-cell receptor (BCR) sequences comprising:
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(A) incubating a plurality of B-cells with a phage antigen display library; (B) distributing the B-cells bound to antigens of the antigen library into a plurality of aliquots; (C) isolating mRNA from B-cells bound to antigens of the antigen library and nucleic acids from the phage; (D) for each aliquot, reverse transcribing mRNA comprising rearranged CDR3 regions of the B-cells using oligonucleotide reverse transcription primers that direct incorporation of an oligonucleotide barcode and a universal adapter resulting in cDNA from each of the light and heavy chain sequences comprising a barcode and a universal adaptor, wherein each of the oligonucleotide reverse transcription primers that are contacted with the contents of a single aliquot share a common barcode sequence; (E) amplifying the light and heavy chain cDNA sequences using amplification primers to obtain amplification products; (F) quantitatively sequencing the amplification products of (E) to obtain a data set of sequences that includes the B-cell light and heavy chain sequences and associated barcodes for each aliquot; (G) sorting amplification products based on the unique barcode to identify light and heavy chain sequences that were amplified from the same aliquot and determining an aliquot occupancy pattern for each unique light and heavy chain sequence; (H) identifying light and heavy chain sequences as paired immune receptor chains based on whether the sequences occur together or do not occur together in a plurality of aliquots based on a statistical probability of observing said aliquot occupancy pattern; (I) generating a library of amplicons by performing PCR on the isolated nucleic acids from the phage, followed by sequencing the library of amplicons; and (J) identifying the paired immune receptor chains in (H) and the nucleic acids in (I) based on whether the sequences occur together or do not occur together in a plurality of aliquots. - View Dependent Claims (26, 27, 28, 29, 30)
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Specification