Abstract 6040: Exploring the impact of analytical factors on B-cell receptor profiling with high-throughput sequencing technologies

Abstract This abstract reflects the views of the authors and should not be construed to represent FDA’s views or policies. High-throughput sequencing of B-cell receptor (BCR) gene rearrangements, along with downstream analysis, provides researchers with the tools to define the B-cell clonal landscape and identify biomarkers for minimal residual disease (MRD). Despite significant advancements in the field over the past decade, comprehensive comparisons on different analytical methods, sequencing technologies, and bioinformatics tools remain largely unexplored. To bridge this gap, the FDA-led B-cell Receptor Sequencing Quality Control (BCR-SEQC) consortium developed protocols for generating reference materials derived from nine well-characterized B-cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors. This study aimed to evaluate the impact of key analytical factors—including input material quality and quantity, sequencing depth, sequencing technologies, and bioinformatics workflows—on BCR sequencing (BCR-seq) performance. The study employed multiple commercial BCR-seq products to prepare libraries with varying input amounts (25ng, 100ng, and 500ng) of reference materials. These libraries were sequenced using three different platforms: Illumina NextSeq 2000, ElementBio Aviti, and MGI DNBSEQ. Subsequently, four bioinformatics workflows (MiXCR, Immcantation, TRUST4, and IGX-Profile) were applied to assemble BCR sequences and determine clonotypes. This research provided the first comprehensive analysis of how variables in wet-lab and computational workflows influence sequencing-based BCR profiling, specifically in terms of linearity, accuracy, and sensitivity. Additionally, the study developed quality control (QC) metrics to ensure the robustness of assays and analytical workflows. Ultimately, this work highlights current capabilities and limitations in BCR-seq technology, identifies critical technical challenges, and establishes actionable best practices for reconstructing BCR repertoires from high-throughput sequencing data. Citation Format: Xiashiyao Zhang, Rittika Mallik, Miguel Pacheco, Shaveta Goyal, Mark Izraelson, Li Song, Henk-Jan van den Ham, Jun Wan, Wenming Xiao, BCR-SEQC consortium. Exploring the impact of analytical factors on B-cell receptor profiling with high-throughput sequencing technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6040.