Terminator Upgrades Single-Cell RNA Sequencing Technology

Japanese scientists have developed a new method for DNA amplification and sequencing that improves the accuracy of single-cell RNA sequencing (scRNA-seq). The new method called TAS-Seq uses a terminal transferase enzyme that amplifies and sequences DNA complementary to mRNA (cDNA) in solid phase on a nanowell/magnetic bead-based cell isolation platform. The use of a dideoxynucleotide triphosphate (ddNTP) terminates the sequencing reaction catalyzed by the terminal transferase and helps reduce variations and handing errors.

The findings were published in an article in the journal Communications Biology, titled “TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq.” In addition to Kouji Matsushima, PhD, an assistant professor at the Tokyo University of Science.

The new method claims higher precision in determining cellular composition in tissues, sensitivity of gene detection, robustness of detecting intercellular interactions, detection of soluble factors, and ease of handling due to the use of a sequence terminator. This technical advancement that uses simple materials and equipment could provide new insights into diseases and drug targets and trigger advances in spatial and single-cell multiomics approaches.

Single-cell studies enable opportunities to understand the complexity of multicellular, heterogenous biological systems at a deeper level that is inaccessible through bulk analyses that provide average cellular readouts. Existing technologies such as droplet- and plate-based scRNA-seq affect the composition of single-cell samples and are restricted by limitations in cDNA amplification that introduce sampling bias and skew scRNA-seq datasets.

The authors note, “TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods.”

The authors compared TAS-Seq and flow-cytometry based scRNA-seq data using lung samples from mice and humans, and found good correlations. In addition to improved gene-detection sensitivity, the researchers detected robust cell-cell interactions and the expression of more growth factors and interleukins in cells using TAS-Seq compared to widely-used, existing scRNA-seq technologies such as 10X Chromium v2 and Smart-seq2 that depend on than template-switching. The authors found TAS-Seq could not only detect more genes, but also identify more genes exhibiting highly variable expressions, compared to other scRNA-seq platforms.

Shichino said, “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. We can detect genes across a wide range of expression levels more uniformly and also detect growth factor and interleukin genes more robustly.”

Based on their findings in lung samples, the authors note, “Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.”

First author of the paper, Shigeyuki Shichino, PhD, an assistant professor at Tokyo University of Science, said “Our technique, TAS-Seq, combines genetic detection sensitivity, robustness of reaction efficiency, and accuracy of cellular composition to enable us to capture important cellular information,”

Unlike polymerase used to amplify DNA, terminal transferase does not require a template. However, it is difficult to handle. The investigators have overcome this challenge by using a ddNTP that terminates the DNA amplification reaction.

Shichino said, “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.”

The new method also decreased cell sampling bias and increases the accuracy of cell composition data though the isolation of single cells using a nanowell/bead-based scRNA-seq platform.

Another advantage of TAS-Seq is its reduced susceptibility 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 said, “We have already completed development of TAS-Seq2, an improved, extensively-optimized version of TAS-Seq. TAS-Seq2 has 1.5 to 2 times more sensitive gene detection in mouse spleen cells.” The researchers at the Tokyo University of Science have established a venture company (ImmunoGenetics) that will provide scRNA-seq services using the new method.