Molecule template estimation using Validator Barcodes as an alternative to UMIs in multiplex PCR for adaptive immune repertoire profiling

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. Human or mouse PBMCs are analyzed by multiplex PCR using V-gene forward primer and J or C gene reverse primers. The libraries either contain VBC or UMI sequences.

T细胞受体（T-cell receptor, TCR）与B细胞受体（B-cell receptor, BCR）测序（简称TCR/BCR-seq）是研究外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）或肿瘤等样本免疫组库的两项重要技术。这类检测的主流方案，是通过靶向V(D)J区域及恒定区的引物对组库进行多重聚合酶链反应扩增，再结合下一代测序（Next-Generation Sequencing, NGS）完成检测。
该检测产生的数据可提供免疫组库相关的多维度信息，包括关键克隆型的存在情况、组库多样性、可变区（V）基因使用偏好、公共克隆型分析等。
在TCR/BCR-seq数据生成过程中，常出现的一个问题是PCR或NGS步骤中产生的序列偏倚。为解决这一问题，研究人员已采用唯一分子标识符（Unique Molecular Identifiers, UMIs）来识别并消除序列偏倚。然而，UMI片段通常较长且序列多样性极高，会导致在多重PCR过程中，UMI序列与大量引物发生非特异性结合，干扰扩增反应。
本研究引入了验证条形码（Validator Barcodes, VBCs），即一套包含8条短条形码的序列，每条长度为6~9个核苷酸。这套精简的条形码序列可提升PCR扩增效率，同时简化PCR引物的设计流程。此外，与UMI类似，VBCs也可用于估算模板分子（RNA或DNA）的数量。
使用标记VBC的引物对PBMC来源的TCR与BCR组库进行谱型分析，得到的结果与基于UMI的检测方法所获结果高度一致，且模板分子计数结果也相仿。总体而言，在TCR与BCR组库检测中，VBCs是一种比UMIs更简便且实用的替代方案。
本研究采用靶向V基因的正向引物与靶向J或C基因的反向引物，通过多重PCR对人源或小鼠源PBMCs进行分析，所制备的文库均包含VBC或UMI序列。