Journal Article

Utilizing Nanopore direct RNA sequencing of blood from patients with sepsis for discovery of co- and post-transcriptional disease biomarkers

He, Jingni, Trevor, Sharon L., Schlapbach, Luregn J., Zhang, Jianshu, Coin, Lachlan J. M., Ganesamoorthy, Devika, McPherson, Stephen J., Gibbons, Kristen S., Kling, Jessica C., Blumenthal, Antje, Chang, Jessie J.-Y.

2025

Overview

Background RNA sequencing of whole blood has been increasingly employed to find transcriptomic signatures of disease states. These studies traditionally utilize short-read sequencing of cDNA, missing important aspects of RNA expression such as differential isoform abundance and poly(A) tail length variation. Methods We used Oxford Nanopore Technologies sequencing to sequence native mRNA extracted from whole blood from 12 patients with definite bacterial and viral sepsis and compared with results from matching Illumina short-read cDNA sequencing data. Additionally, we explored poly(A) tail length variation, novel transcript identification, and differential transcript usage. Results The correlation of gene count data between Illumina cDNA- and Nanopore RNA-sequencing strongly depended on the choice of analysis pipeline; NanoCount for Nanopore and Kallisto for Illumina data yielded the highest mean Pearson’s correlation of 0.927 at the gene level and 0.736 at the transcript isoform level. We identified 2 genes with differential polyadenylation, 9 genes with differential expression and 4 genes with differential transcript usage between bacterial and viral infection. Gene ontology gene set enrichment analysis of poly(A) tail length revealed enrichment of long tails in mRNA of genes involved in signaling and short tails in oxidoreductase molecular functions. Additionally, we detected 240 non-artifactual novel transcript isoforms. Conclusions Nanopore RNA- and Illumina cDNA-gene counts are strongly correlated, indicating that both platforms are suitable for discovery and validation of gene count biomarkers. Nanopore direct RNA-seq provides additional advantages by uncovering additional post- and co-transcriptional biomarkers, such as poly(A) tail length variation and transcript isoform usage.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Bacterial infections / Bias / Biological markers / Biomarkers / Biomarkers - blood / Blood / Correlation / Correlation analysis / Datasets / Diagnosis / Differential transcript usage / Direct RNA-sequencing / Female / Gene expression / Gene Expression Profiling / Gene sequencing / Gene set enrichment analysis / Genes / Genetic transcription / Health aspects / Humans / Identification and classification / Infections / Infectious Diseases / Internal Medicine / Isoforms / Long-read sequencing / Male / Medical examination / Medical Microbiology / Medicine / Medicine & Public Health / Middle Aged / Nanopore Sequencing - methods / Nanopores / Novel isoform detection / Oxford Nanopore Technologies / Oxidoreductase / Parasitology / Polyadenine / Polyadenylation / Post-transcription / Ribonucleic acid / RNA / RNA sequencing / RNA, Messenger - blood / RNA, Messenger - genetics / Sepsis / Sepsis - blood / Sepsis - diagnosis / Sepsis - genetics / Sequence Analysis, RNA - methods / Software / Transcriptome / Transcriptomics / Tropical Medicine / Variation / Viral infections