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Chris Carlson, Ryan Emerson, Anna Sherwood, Cindy Desmarais, Moon Chung, Joe Parsons, Marissa LaMadrid-Herrmannfeldt, Robert Livingston, Mark Rieder, Harlan Robins, Minimizing PCR bias in high-throughput adaptive immune repertoire sequencing assays (P3389), The Journal of Immunology, Volume 190, Issue Supplement_1, May 2013, Page 135.25, https://doi.org/10.4049/jimmunol.190.Supp.135.25
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Abstract
The human adaptive immune system is a primary defense against the vast world of potential pathogens. To counter this, B and T cells rearrange a diverse repertoire of surface receptors. This system functions in part by clonal expansion, so measuring changes in the abundance of both unique and total clones is necessary to understand the immune response. We developed immune profiling assays to amplify and sequence rearranged receptors. To efficiently capture receptor diversity we use a multiplex PCR approach. However, small variations in annealing kinetics can have large impacts on amplification efficiency causing PCR amplification bias (PAB). To address this issue, we developed a method to quantify and correct PAB introduced by multiplex PCR. We demonstrate this method using our T cell receptor γ (TCRG) assay. Our method uses a quantitated, synthetic immune repertoire standard consisting of every possible Variable(V) and Joining(J) gene combination. Following multiplex TCRG PCR amplification, we assessed the difference between the synthetic VJ composition before and after amplification and quantified PAB. We adjusted primer concentrations and redesigned primers to generate an unbiased multiplex PCR assay. Using this method we reduced PAB for the TCRG assay from a 340-fold to a 4.5-fold dynamic range. Now the abundance of receptor sequences is representative of the frequency of each receptor chain in the input sample, allowing for accurate quantification of the TCRG repertoire.
