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

BMC Genomics. 2020 Jul 2;21(1):456. doi: 10.1186/s12864-020-06843-0.


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.

Keywords: Exclusion amplification; Index hopping; Multiplexing; Next-generation sequencing; NovaSeq; Single-cell RNA sequencing; TruSeq; inDrop.

MeSH terms

  • Animals
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Mice
  • Sequence Alignment
  • Sequence Analysis, RNA / methods*
  • Sequence Analysis, RNA / standards
  • Single-Cell Analysis / methods*
  • Single-Cell Analysis / standards