Using synthetic templates to design an unbiased multiplex PCR assay

Christopher S. Carlson, Ryan O. Emerson, Anna M. Sherwood, Cindy Desmarais, Moon-Wook Chung, Joseph M. Parsons, Michelle S. Steen, Marissa A. LaMadrid-Herrmannsfeldt, David W. Williamson, Robert J. Livingston, David Wu, Brent L. Wood, Mark J. Rieder and Harlan Robins ()
Additional contact information
Christopher S. Carlson: Fred Hutchinson Cancer Research Center
Ryan O. Emerson: Adaptive Biotechnologies
Anna M. Sherwood: Adaptive Biotechnologies
Cindy Desmarais: Adaptive Biotechnologies
Moon-Wook Chung: Adaptive Biotechnologies
Joseph M. Parsons: Adaptive Biotechnologies
Michelle S. Steen: Adaptive Biotechnologies
Marissa A. LaMadrid-Herrmannsfeldt: Adaptive Biotechnologies
David W. Williamson: Adaptive Biotechnologies
Robert J. Livingston: Adaptive Biotechnologies
David Wu: University of Washington
Brent L. Wood: University of Washington
Mark J. Rieder: Adaptive Biotechnologies
Harlan Robins: Fred Hutchinson Cancer Research Center

Nature Communications, 2013, vol. 4, issue 1, 1-9

Abstract: 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.

Date: 2013
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DOI: 10.1038/ncomms3680

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