title: Using synthetic templates to design an unbiased multiplex PCR assay.
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.
authors.affiliation: 1] Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington 98109, USA [2].
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Carlson
authors.forename: Ryan O
authors.initials: RO
authors.lastname: Emerson
authors.forename: Anna M
authors.initials: AM
authors.lastname: Sherwood
authors.forename: Cindy
authors.initials: C
authors.lastname: Desmarais
authors.forename: Moon-Wook
authors.initials: MW
authors.lastname: Chung
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Parsons
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Steen
authors.forename: Marissa A
authors.initials: MA
authors.lastname: LaMadrid-Herrmannsfeldt
authors.forename: David W
authors.initials: DW
authors.lastname: Williamson
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Livingston
authors.forename: David
authors.initials: D
authors.lastname: Wu
authors.forename: Brent L
authors.initials: BL
authors.lastname: Wood
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Rieder
authors.forename: Harlan
authors.initials: H
authors.lastname: Robins
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D017931:DNA Primers; D015342:DNA Probes / Q000737:chemistry*; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D016130:Immunophenotyping; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D011947:Receptors, Antigen, B-Cell / Q000145:classification / Q000235:genetics* / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000145:classification / Q000235:genetics* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D011947:Receptors, Antigen, B-Cell; D011948:Receptors, Antigen, T-Cell