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

Abstract

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.

Nonmuscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli

“…, 2011 ), our analysis of recently generated single-cell RNAseq (scRNAseq) data from the intestinal tract ( Banerjee et al. , 2020 ; Southard-Smith et al. , 2020 ) revealed that NM2C is preferentially expressed in mature villus enterocytes relative to NM2A or NM2B, the latter of which was barely detectable (Supplemental Figure 1).…”

Microfluidics applications for high-throughput single cell sequencing

“…The hydrogel beads dissolve in the droplets, releasing the oligonucleotides to hybridize the mRNAs, and the reverse transcription reactions are carried out within the drops. However, the major drawback of inDrop is the extremely low cell capture efficiency, which could detect only 20–50 copies of transcripts per cell [ 118 ]. This technology enables the scRNA-seq of large numbers of cells, which allows the identification of very rare cell types from heterogeneous populations.…”

A Primer for Single-Cell Sequencing in Non-Model Organisms

“…However, Drop-seq requires custom, “lab-made” microfluidics devices which requires technical engineering expertise [ 37 ]. InDrop has a commercially available platform, but the cell and transcript capture efficiency is low [ 38 ]. These microfluidics scRNA-seq approaches are beneficial because the RNA extraction and library preparation can occur within the microfluidic chip; however, these approaches can be problematic because they require either technical expertise or they are not sensitive to the distribution of transcripts and cells in a sample.…”