Researchers are taking RNA sequencing to the next level to better understand sepsis, a serious condition caused by the body’s extreme response to infection. A team led by researchers a from the University of Melbourne used the Oxford Nanopore Technologies platform to directly sequence RNA from the blood of 12 patients with bacterial and viral sepsis. Unlike traditional methods that require converting RNA into cDNA and often miss important information, this direct approach captures the full complexity of RNA molecules. By comparing results from Nanopore sequencing with standard Illumina short-read cDNA sequencing, the team found that both methods gave highly similar gene count data—especially when using optimized analysis tools. But Nanopore sequencing went further, revealing differences in the length of poly(A) tails (which affect RNA stability), identifying new transcript variants, and detecting subtle shifts in how genes are used depending on whether the patient had a bacterial or viral infection.

Poly(A) length distribution and Gene Set Enrichment Analysis (GSEA)
using genes ranked by poly(A) tail lengths

A Poly(A) length distribution in mitochondrial transcripts. B Poly(A) length distribution in nuclear transcripts. C-E Ridgeplots from the clusterProfiler package with the X-axis indicating the poly(A) lengths and Y-axis indicating the GO term or KEGG pathway. The distribution is the distribution of poly(A) length of those genes that enriched in the corresponding GO enrichment analysis C molecular function D cellular component and E KEGG pathway, and the colour indicates the significance, with adjusted P-value < 0.05 deemed as significant (the full list of significant pathways can be viewed in Supplementary Tables 2–4). The mitochondrial transcripts are excluded and numbers on the plots indicate the number of genes relevant to the GO term/pathway

They discovered:

• 2 genes with changes in poly(A) tail length
• 9 genes that were expressed differently between infection types
• 4 genes with differences in transcript usage
• 240 novel transcript isoforms not previously recorded

Interestingly, genes involved in signaling had longer poly(A) tails, while those involved in oxidation-reduction reactions had shorter ones. These molecular signatures could serve as new biomarkers, improving the way we diagnose and understand sepsis.

This work highlights how direct RNA sequencing can uncover deeper layers of gene expression that might be missed by conventional methods, offering powerful insights into diseases where timing and precision matter.