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Molecule template estimation using Validator Barcodes as an alternative to UMIs 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). Very similar analytic results and minimal template values have been generated when using VBC-labeled primers in TCR and BCR repertoire profiles from PBMCs to those generated by UMI-based assay counts. Overall, VBCs are a useful and simpler alternative to UMIs in assaying TCR and BCR repertoires.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE297422 | GEO | 2025/05/16

REPOSITORIES: GEO

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