Abstract 4073: Massively parallel barcoding as an alternative to genetic demultiplexing in large cohort scRNA-Seq studies

Abstract Single-cell RNA sequencing (scRNA-Seq) studies with large sample cohorts are expensive, and prone to batch effects. Genetic demultiplexing allows for pooling of distinct samples beforehand and then using bioinformatic tools post sequencing to identify individual samples based on their genetic makeup. This allows for super-loading conventional scRNA-seq runs while reducing costs and batch effects. However, these strategies often lead to up to 30% doublets and are limited by the quality of the reference genome used, and the genetic diversity of the input. Additional bulk sequencing and computational analyses add on costs and times to identify individual samples. This problem becomes more acute in diseases with heavy mutational burden like cancer - tumor-driven mutations might overshadow natural genetic variation between samples and extra sequencing and analyses might be warranted. Scale Bio has pioneered the use of massively parallelized barcoding and novel multiplexing strategies (ScalePlex) to boost throughput and circumvent the need for additional sample demultiplexing strategies to identify individual samples. Typical doublet rates in a Scale Bio scRNA-Seq library are around 5% and the protocol allows for simultaneous profiling of up to thousands of samples with Scaleplex, with significantly lower per sample inputs and no additional bulk sequencing required. Here we demonstrate the utility of the Scale Bio scRNA-Seq with and without Scaleplex in sample demultiplexing 12 samples from diseased and non-diseased individuals with similar genetic backgrounds when compared to super loading a conventional scRNA-seq run. Furthermore, we compare our data with current on market, publicly available computational tools for genetic demultiplexing with ScalePlex readouts as the ground truth. We observe low doublet rates in the Scale Bio data along with maximum utilization of possible sequencing reads, and clear, unambiguous sample identification when compared to conventional scRNA-seq techniques and analyses tools. Thus, the Scale Bio scRNA-seq platform offers a viable, low cost and effort platform for conventional genetic demultiplexing techniques with better resolution and higher sample multiplexing capacity. Furthermore, ScalePlex provides an alternative to easily, distinctly barcode and multiplex samples without genetic demultiplexing where researchers can gain the same benefits of reduced cost and batch effects while removing sample heterogeneity considerations and bioinformatics barrier of genetic demultiplexing. Citation Format: Sanika Khare, Dominic Skinner, Austin Holmes, Felix Schlesinger, Keith Jackson. Massively parallel barcoding as an alternative to genetic demultiplexing in large cohort scRNA-Seq studies [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 4073.