Molecule template estimation using Validator Barcodes in multiplex PCR for adaptive immune repertoire profiling

Abstract

TCR- and BCR-sequencing (TCR/BCR-seq) are two important technologies in studying the immune repertoire of samples such as PBMCs or tumors. In their most common form, these assays combine multiplex PCR of the repertoire using primers targeting regions of the V(D)J and the constant region with next-generation sequencing (NGS). The data produced by this assay provide a slew of information regarding immune repertoire(s) including the presence critical clonotypes, repertoire diversity, variable (V) gene usage, analysis of public clonotypes, etc. One issue that can arise during generation of the TCR/BCR-seq data is sequence bias during the PCR or NGS steps. To combat this, unique molecular identifiers (UMIs) have been used to identify and eliminate sequence bias. However, UMI fragments can be long and very diverse, resulting in the UMI sequences interfering with any of the multitude of primers during multiplex PCR. Here, we introduce Validator Barcodes (VBCs), a set of eight short barcodes (6-9 nucleotides in length). This compact set of barcodes improves PCR efficiency and facilitates PCR primer designs. Also, like UMIs, the VBCs may be used to estimate the number of template molecules (RNA or DNA). Using VBC-labeled primers for TCR and BCR repertoire profiling from PBMCs produces highly comparable results and similarly template values to those obtained through UMI-based assay counts. Overall, VBCs are a useful and simpler alternative to UMIs in assaying TCR and BCR repertoires.