New scRNA-seq Tool Overcomes Accuracy Issues

A research team in Japan has developed a new and improved technique for single-cell RNA sequencing (scRNA-seq) that uses simple materials and equipment to provide higher-precision data than current, widely used technologies.

scRNA-seq has revolutionized medicine and biology by providing the ability to study the inner workings of thousands of cells at one go.  But traditional scRNA-seq methods are limited by potential inaccuracies in determining cell composition and inefficient complementary DNA (cDNA) amplification—a process by which a double-stranded DNA that ‘complements’ the single-stranded RNA is generated and replicated millions of times—by the commonly-used template-switching reaction.

Dubbed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq), the new method provides genetic detection sensitivity, robustness of reaction efficiency, and accuracy of cellular composition. It uses a template independent enzyme for cDNA amplification called terminal transferase (TdT). TdT can be difficult to handle, but the research team included dideoxynucleotide phosphate (ddNTP) as a ‘terminator’ for the cDNA amplification reaction to overcome this.

“ddNTP spike-in, specifically dideoxycytidine phosphate (ddCTP), stops the excessive extension of polyN-tail by TdT in a stochastic manner, and greatly reduces the technical difficulties of the TdT reaction,” says Assistant Prof. Shigeyuki Shichino of the Tokyo University of Science. 

TAS-Seq also uses a nanowell/bead-based scRNA-seq platform, which allows the isolation of single cells in tissue samples, thereby decreasing cell sampling bias and improving the accuracy of cell composition data.

To verify the efficiency of TAS-Seq, Shichino and colleagues compared it to the current, widely used scRNA-seq techniques, 10X Chromium V2 and Smart-seq2, using murine and human lung tissue samples. They found that TAS-Seq could not only detect more genes overall, but also identify more highly variable genes, when compared to major scRNA-seq platforms. The findings were published recently in the journal Communications Biology.

 “We found that TAS-Seq may outperform 10X Chromium V2 and Smart-seq2 in terms of gene detection sensitivity and gene drop-out rates, indicating that TAS-Seq might be one of the most sensitive high-throughput scRNA methods,” says Shichino. “We can detect genes across a wide range of expression levels more uniformly and also detect growth factor and interleukin genes more robustly.”

An added advantage of the new method is that TAS-Seq is less susceptible to batch effects. TAS-Seq data was also highly correlated with flow-cytometric data on the tissue samples, indicating that it can generate highly accurate cell composition data.

Shichino says the team has already completed development of TAS-Seq2, “an improved, extensively-optimized version of TAS-Seq” with 1.5 to 2 times more sensitive gene detection in mouse spleen cells. They have also established ImmunoGenetics, a venture company from Tokyo University of Science, to provide scRNA-seq services using TAS-Seq and TAS-Seq2.

The development of TAS-Seq and TAS-Seq2 will lead to the discovery of new therapeutic targets for diseases and advancements in the field of spatial transcriptomics, which also relies on solid-phase cDNA synthesis. It will also accelerate the development of single-cell omics technology, thereby promoting the understanding of biology and disease development and progression.