Anchor-Enhanced Bead Design for Reduced Oligonucleotide Synthesis Errors in Single-cell sequencing

Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a 'V' base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods. In order to evaluate the accuracy of our spacer UMI assignment we captured mixed mouse and human cells (at ratio of 50:50) and then performed single-cell RNA sequencing.

单细胞转录组学（single-cell transcriptomics）依赖将条形码（barcodes）与唯一分子标识符（unique molecular identifiers, UMIs）整合至捕获的聚腺苷酸化mRNA（polyA+ mRNA）中，但其发展面临一项重大挑战：寡核苷酸捕获序列的合成误差。这类误差在长读长测序（long-read sequencing）场景中尤为突出，会干扰序列的精准识别，并导致UMI去重（UMI deduplication）结果出现偏差。为缓解该问题，本研究对寡核苷酸捕获设计进行了优化：在条形码与UMI之间嵌入一段锚定序列，并在polyA捕获区域旁侧添加一个“V”型碱基锚定序列。该配置方案可同时兼容短读长测序（short-read sequencing）与长读长测序技术，有助于提升UMI回收率与特征检测效能，进而优化基于液滴的测序方法的整体性能。为验证我们的间隔区UMI分配方案的准确性，本研究以50:50的比例混合捕获小鼠与人类细胞，并开展了单细胞RNA测序实验。