Epithelial Biology Center and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA.
BMC Genomics. 2020 Jul 2;21(1):456. doi: 10.1186/s12864-020-06843-0.
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.
单细胞 RNA 测序（scRNA-seq）实验的需求不断增加，例如每个实验的实验数量和查询的细胞数量，这需要更高的测序深度和更高的数据质量。新型高通量测序仪，如 Illumina NovaSeq 6000，能够以经济有效的方式满足这一需求。然而，当前的 scRNA-seq 文库设计与新型测序技术（如索引跳跃）不兼容，并且它们生成高质量数据的能力尚未得到系统评估。
在这里，我们在 inDrop scRNA-seq 平台的基础上设计了一种双索引文库结构，称为 TruDrop，以解决这些兼容性挑战，使得 TruDrop 文库和标准的 Illumina 文库可以在 NovaSeq 上同时进行测序。在 scRNA-seq 文库上，我们实施了一种先前记录的针对索引跳跃这一众所周知问题的对策，在 NovaSeq 上显著提高了碱基调用的准确性，并提供了同时多路复用 24 个 scRNA-seq 文库的示例。与先前的 inDrop 文库相比，我们展示了 TruDrop 在转录多样性方面的有利比较。
我们的方法能够以经济高效的方式生成高质量的测序数据，这应该能够更常规地使用 scRNA-seq 技术。
