Abstract
T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.
MeSH terms
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B-Lymphocytes / immunology
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B-Lymphocytes / metabolism
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B-Lymphocytes / pathology
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DNA Primers
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DNA Probes / chemistry*
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Gene Expression
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Gene Library
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Humans
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Immunophenotyping
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Multiplex Polymerase Chain Reaction / methods*
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Multiplex Polymerase Chain Reaction / standards
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Neoplasm, Residual / diagnosis
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Neoplasm, Residual / genetics*
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Neoplasm, Residual / immunology
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Neoplasm, Residual / pathology
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / diagnosis
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / genetics*
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / immunology
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Precursor Cell Lymphoblastic Leukemia-Lymphoma / pathology
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Receptors, Antigen, B-Cell / classification
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Receptors, Antigen, B-Cell / genetics*
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Receptors, Antigen, B-Cell / immunology
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Receptors, Antigen, T-Cell / classification
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Receptors, Antigen, T-Cell / genetics*
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Receptors, Antigen, T-Cell / immunology
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T-Lymphocytes / immunology
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T-Lymphocytes / metabolism
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T-Lymphocytes / pathology
Substances
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DNA Primers
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DNA Probes
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Receptors, Antigen, B-Cell
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Receptors, Antigen, T-Cell