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

• Published in: BMC genomics Vol. 21; no. 1; p. 456
• Main Authors: Southard-Smith, Austin N, Simmons, Alan J, Chen, Bob, Jones, Angela L, Ramirez Solano, Marisol A, Vega, Paige N, Scurrah, Cherie' R, Zhao, Yue, Brenan, Michael J, Xuan, Jiekun, Shrubsole, Martha J, Porter, Ely B, Chen, Xi, Brenan, Colin J H, Liu, Qi, Quigley, Lauren N M, Lau, Ken S
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 02.07.2020; BioMed Central; BMC
• Subjects: Accuracy; Animals; Biotechnology industries; Compatibility; Gene sequencing; Genomics; High-Throughput Nucleotide Sequencing; Humans; Index hopping; inDrop; Libraries; Methodology; Mice; Multiplexing; Next-generation sequencing; NovaSeq; Quality; Ribonucleic acid; RNA; RNA sequencing; Sequence Alignment; Sequence Analysis, RNA - methods; Sequence Analysis, RNA - standards; Single-Cell Analysis - methods; Single-Cell Analysis - standards; Single-cell RNA sequencing; Transcription; TruSeq; United States; Multiplexing; Exclusion amplification; Next-generation sequencing; Single-cell RNA sequencing; Index hopping; TruSeq; NovaSeq; inDrop
• ISSN: 1471-2164; 1471-2164
• 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.