The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

中文翻译：

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双索引文库设计使In-Drop单细胞RNA测序与exAMP化学测序平台兼容。

单细胞RNA测序（scRNA-seq）实验的需求不断增长，例如实验数量和每个实验查询的细胞数量，需要更高的测序深度和更高的数据质量。新型的高通量测序仪，例如Illumina NovaSeq 6000，可以以经济高效的方式满足这一需求。但是，当前的scRNA-seq文库设计与较新的测序技术（如索引跳跃）提出了兼容性挑战，并且其生成高质量数据的能力尚未得到系统的评估。在这里，我们在inDrop scRNA-seq平台之上设计了一个双索引的库结构，称为TruDrop，以解决这些兼容性挑战，从而使TruDrop库和标准Illumina库可以在NovaSeq上彼此并排测序。在scRNA-seq库中，我们针对已知的索引跳跃问题实施了先前记录的对策，在NovaSeq上证明了碱基调用准确性的显着提高，并提供了同时复用24个scRNA-seq库的示例。与先前的inDrop文库相比，我们在TruDrop的转录多样性上显示了有利的比较。我们的方法能够以高成本效益，高通量生成高质量的测序数据，这将使更多的例行使用scRNA-seq技术成为可能。与先前的inDrop文库相比，我们在TruDrop的转录多样性上显示了有利的比较。我们的方法能够以高成本效益，高通量生成高质量的测序数据，这将使更多的例行使用scRNA-seq技术成为可能。与先前的inDrop文库相比，我们在TruDrop的转录多样性上显示了有利的比较。我们的方法能够以高成本效益，高通量生成高质量的测序数据，这将使更多的例行使用scRNA-seq技术成为可能。