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Dual indexed design of in-Drop single-cell RNA-seq libraries improves sequencing quality and throughput

By Austin N Southard-Smith, Alan J. Simmons, Bob Chen, Angela L. Jones, Marisol A. Ramirez Solano, Paige N Vega, Cherie’ R. Scurrah, Yue Zhao, Michael J Brenan, Jiekun Xuan, Ely B Porter, Xi Chen, Colin J.H. Brenan, Qi Liu, Lauren N.M. Quigley, Ken Lau

Posted 08 Nov 2019
bioRxiv DOI: 10.1101/835488 (published 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 new 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. We overcame the index-hopping issue, demonstrated significant improvements in base-calling accuracy, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior library structures. Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

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