New sensitive and robust single-cell RNA sequencing technique outperforms competition

The new technique uses a nanowell/magnetic bead-based single-cell RNA sequencing platform, terminal transferase enzyme and dideoxynucleotide phosphate terminators to generate highly accurate information about gene expression, cell composition and cell-cell interactions. Credit: Tokyo University of Science

The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized medicine and biology by providing the opportunity to study the inner workings of thousands of cells at once. But scRNA-seq methods are limited by potential inaccuracies in determining cell composition and inefficient complementary DNA amplification (cDNA) – a process in 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 professor Shigeyuki Shichino and Prof. Kouji Matsushima of Tokyo University of Science, 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 deliver scRNA-seq data with higher precision than currently widely used technologies.

“Our technique, TAS-Seq, combines genetic detection sensitivity, robustness of response efficiency and accuracy of cellular composition to allow us to capture important cellular information,” said assistant professor Shichino. The study is published in Communication 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.






The new solid-phase (TAS-Seq) terminator-assisted complementary DNA amplification and sequencing method (TAS-Seq) provides highly accurate data on gene expression. Credit: Tokyo University of Science

TAS-Seq uses a template-independent enzyme for cDNA amplification called terminal transferase (TdT). But TdT is difficult to handle. To overcome 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 elongation of polyN tail by TdT in a stochastic manner and significantly reduces the technical difficulties of the TdT reaction,” explains assistant professor Shichino.

TAS-Seq also utilizes a nanowell/bead-based scRNA-seq platform, which facilitates the isolation of single cells in tissue samples, thereby reducing the preference for cell sampling and improving the accuracy of cell composition data. The research team then verified the efficiency of TAS-Seq and compared it to the currently widely used scRNA-seq techniques, 10X Chromium V2 and Smart-seq2, using mouse and human lung tissue samples. They found that TAS-Seq could not only detect more genes overall, but also identify more highly variable genes, compared to large scRNA-seq platforms.

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

An additional advantage of the new method is that TAS-Seq is less sensitive 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 of the future, Assistant Professor Shichino says: “We have already completed development of TAS-Seq2, an enhanced, extensively optimized version of TAS-Seq. TAS-Seq2 has 1.5 to 2 times more sensitive gene detection in the spleen from mice cellsThe research team also established ImmunoGenetics, a Tokyo University of Science venture, to provide scRNA-seq services using TAS-Seq and TAS-Seq2.

scRNA-seq is an important tool for medical and biological researchers. The development of TAS-Seq and TAS-Seq2 will lead to the discovery of new therapeutic targets for diseases and advances in “spatial transcriptomics”, which also rely on solid phase cDNA synthesis. It will also accelerate the development of single-cell omics technology, furthering our understanding of the principles of biology and disease development and progression.


Benchmarking of Transcriptomics Integration Methods for Prediction of Transcript Distribution and Cell Type Deconvolution


More information:
Shigeyuki Shichino et al, TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq, Communication biology (2022). DOI: 10.1038/s42003-022-03536-0

Quote: New sensitive and robust single-cell RNA sequencing technique outperforms competition (2022, June 27) retrieved June 27, 2022 from https://phys.org/news/2022-06-sensitive-robust-single-cell-rna-sequencing . html

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