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Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms


Southard-Smith ANAustin N , Simmons AJAlan J , Chen BBob , Jones ALAngela L , Ramirez Solano MAMarisol A , Vega PNPaige N , Scurrah CRCherie' R , Zhao YYue , Brenan MJMichael J , Xuan JJiekun , Shrubsole MJMartha J , Porter EBEly B , Chen XXi , Brenan CJHColin J H , Liu QQi , Quigley LNMLauren N M , Lau KSKen S . BMC genomics. 2020 7 2; 21(1). 456


BACKGROUND: 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.

RESULTS: 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.

CONCLUSIONS: Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.