The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized the fields of medicine and biology by providing the ability to study the inner workings of thousands of cells at one go. But 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.
Recently, a research team from Japan, led by Assistant Prof. Shigeyuki Shichino and Prof. Kouji Matsushima of Tokyo University of Science, has developed a new and improved technique for scRNA-seq. The new method, terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq), uses simple materials and equipment to provide higher-precision scRNA-seq data than current, widely-used technologies. “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,” reveals Assistant Prof. Shichino. The study was published in Communications Biology on June 27, 2022. The research team also included Associate Prof. Satoshi Ueha of Tokyo University of Science, Prof. Taka-aki Sato of the University of Tsukuba, and Prof. Shinichi Hashimoto of Wakayama Medical University.
TAS-Seq uses a template independent enzyme for cDNA amplification called terminal transferase (TdT). But TdT is difficult to handle. To surmount this challenge, the research team included dideoxynucleotide phosphate (ddNTP) as a ‘terminator’ for the cDNA amplification reaction. “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,” explains Assistant Prof. Shichino. 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.
The research team then verified the efficiency of TAS-Seq and 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. Assistant Prof. Shichino says, “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.”
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.
Speaking on the future, Assistant Prof. Shichino reveals, “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 research team has also established ImmunoGenetics, a venture company from Tokyo University of Science, to provide scRNA-seq services using TAS-Seq and TAS-Seq2.
scRNA-seq is an important tool for medical and biology researchers. 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 our understanding of the principles of biology and disease development and progression.
About The Tokyo University of Science
Tokyo University of Science (TUS) is a well-known and respected university, and the largest science-specialized private research university in Japan, with four campuses in central Tokyo and its suburbs and in Hokkaido. Established in 1881, the university has continually contributed to Japan’s development in science through inculcating the love for science in researchers, technicians, and educators.
With a mission of “Creating science and technology for the harmonious development of nature, human beings, and society”, TUS has undertaken a wide range of research from basic to applied science. TUS has embraced a multidisciplinary approach to research and undertaken intensive study in some of today’s most vital fields. TUS is a meritocracy where the best in science is recognized and nurtured. It is the only private university in Japan that has produced a Nobel Prize winner and the only private university in Asia to produce Nobel Prize winners within the natural sciences field.
About Assistant Professor Shigeyuki Shichino and Professor Kouji Matsushima from Tokyo University of Science
Assistant Prof. Shigeyuki Shichino is part of the Research Institute for Biomedical Sciences, Tokyo University of Science. His research focuses on system genome science, including transcriptome, single-cell, and interactome network, and experimental pathology, including lung fibrosis, macrophage/fibroblast biology, and single-cell RNA sequencing. He has published 21 papers.
Prof. Kouji Matsushima is part of the Research Institute for Biomedical Sciences, Tokyo University of Science. His research focuses on inflammation, immunology, and cancer immunotherapy. He was conferred a Lifetime Honorary Membership Award by the International Cytokine and Interferon Society in 2019. In 2021, he won the Takeda Prize for Medical Science.
The study was supported by the Japan Agency for Medical Research and Development PRIME program (JP21gm6210025, Shigeyuki Shichino) and the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research of Innovative Areas Program (Inflammation Cellular Sociology, 17H06392, Kouji Matsushima), and Grant-in-Aid for Young Scientists (19K16620, Shigeyuki Shichino).