Researchers at the University of Hong Kong have developed a powerful new tool that reveals overlooked features in RNA sequencing data. The method, called scTail, was developed to improve our understanding of how genes are regulated at the RNA level, particularly the ends of RNA molecules where polyadenylation sites (PAS) are located.

Polyadenylation sites are important because they help determine the stability, transport, and translation of RNA. In single-cell RNA sequencing, especially the 3′ scRNA-seq method, most researchers focus on second-strand reads, which give information about gene expression. However, first-strand reads (often called “reads 1”) can contain valuable data about PAS, but these are often discarded or trimmed.

The scTail method changes that. It makes use of the first-strand reads to detect PAS locations with high precision and sensitivity. Then, it uses the second-strand reads to measure how often these sites are used. This approach allows researchers to identify alternative polyadenylation, a process where different PAS are used in the same gene, influencing how the gene functions in different biological conditions.

Developing scTail to identify PAS from 3′ tag-based scRNA-seq

A Outline of the first step of scTail. The purpose of this step is to get the position of PAS from aligned bam file. In the real situation, calling peak was going on for each sample (one bam file). Then, peaks of all samples were merged. The red circle is the terminate position of first strand reads. B Schematic of the second step of scTail. An aligned bam file was inputted and a sparse matrix (h5ad file) was obtained as output. C Flowchart of deep neural network embedded into scTail to filter low-quality putative PASs from sequence

By combining scTail with another tool called BRIE2, the researchers could explore how alternative PAS usage changes in diseases like cancer or during biological processes such as development over time. This deeper understanding of RNA regulation opens up new possibilities for diagnostics and treatments.

Availability – scTail is an open-source Python package publicly available at https://github.com/StatBiomed/scTail