Alternative splicing is a process that allows a single gene to produce multiple RNA transcripts, called isoforms. This greatly increases the diversity of proteins that cells can make, and it plays an important role in many biological processes. When alternative splicing is disrupted, it can contribute to diseases such as cancer and neurological disorders.

Traditional sequencing methods, often referred to as short read sequencing, can measure RNA levels very accurately but struggle to capture full-length RNA molecules. This makes it difficult to determine which isoforms are present, especially when transcripts are complex. Long read sequencing technologies help solve this problem by reading entire RNA molecules in one continuous sequence, allowing researchers to directly observe different isoforms.

In this work, led by researchers at the University of Melbourne, researchers describe how long read sequencing provides a clearer view of alternative splicing. By capturing full-length RNA sequences, scientists can better understand how genes are regulated and how different isoforms affect protein function.

Long-read methods for profiling isoforms

(A) An overview of available long-read methodologies for isoform characterisation. A variety of sample inputs (orange boxes) can be used with different sequencing methods (dashed edges) to answer a wide range of experimental questions. Intermediate experiment steps are shown with a bold outline. RNA from bulk samples can be either sequenced directly (ONT direct RNA) or reverse transcribed to cDNA, while RNA from single cells currently needs to be processed into cDNA before sequencing. Once present as cDNA, samples can be sequenced directly without PCR (unamplified cDNA) or after whole-transcriptome PCR amplification (PCR-cDNA) depending on starting amounts. cDNA can also serve as input for target enrichment techniques including PCR for specific gene isoforms of interest (amplicon sequencing) or capture of target cDNAs for sequencing (CaptureSeq). Long-read spatial transcriptomics starts with tissue sections placed on slides covered with spatially barcoded oligonucleotides. Cellular RNA is captured by nearby barcodes allowing generation of spatially barcoded cDNAs. Sequencing identifies spatial expression patterns of RNA isoforms within the tissue such as the hypothetical isoform A. (B) Selected examples where long-read sequencing methods have been used to examine isoforms and alternative splicing in a range of human diseases. AR, androgen receptor; CLL, chronic lymphocytic leukemia; PacBio, Pacific Biosciences; ONT, Oxford Nanopore Technologies.

The approach becomes even more powerful when combined with other advanced methods. For example, pairing long read sequencing with single cell sequencing allows researchers to see how splicing varies from cell to cell. Spatial transcriptomics adds another layer by showing where these isoforms are expressed within tissues. Together, these technologies provide a much more detailed picture of RNA biology.

Another important advance is the ability to connect genetic variation to isoform expression. By integrating sequencing data with genetic information, researchers can begin to understand how specific mutations influence splicing patterns. This is especially important in tissues like the brain, where alternative splicing is highly complex and tightly regulated.

Overall, long read RNA sequencing is helping scientists uncover the full landscape of alternative splicing. These insights may lead to new therapeutic targets and a better understanding of how gene regulation goes wrong in disease.