Innovative Low-cost Probe Generation Empowers Targeted Long-read RNA Sequencing

Alternative splicing of human genes is a cornerstone of genetic complexity and diversity, enabling a single gene to produce many different proteins [1].This is not only crucial for human development but also plays pivotal roles in many diseases [2].Over the years, next-generation sequencing (NGS) has been instrumental in unraveling the intricacies of human gene splicing [3].It has led to significant advancements in our understanding, with notable examples being the identification of splice variants in BRCA1 and BRCA2 genes, which are critical in breast and ovarian cancer risk assessment [4].However, the complexity of human genes, which consist of ten exons on average, poses a challenge for the short-read sequencing approaches commonly used in NGS platforms like Illumina.Specifically, the combinatorial patterns of tens of exons can generate many isoforms, which are very challenging or impossible for short reads (up to 300 bp) to resolve.In contrast, long-read sequencing technologies, such as those offered by Oxford Nanopore Technologies (ONT) and Pacific Biosciences, represent a paradigm shift.Their ability to read several kilobases to tens of kilobases in a single read allows them to capture entire transcripts, end-to-end [5].This has led to groundbreaking discoveries in gene splicing, unveiling a new era of research in human health and disease [6,7].Large-scale studies using these technologies have reported the discovery of tens of thousands of novel isoforms [8].