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Background <p>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.</p> Methods <p>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.</p> Results <p>The correlation of gene count data between Illumina cDNA- and Nanopore RNA-sequencing strongly depended on the choice of analysis pipeline; <i>NanoCount</i> for Nanopore and <i>Kallisto</i> 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.</p> Conclusions <p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月13日

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  • Jingni He,
  • Devika Ganesamoorthy,
  • Jessie J.-Y. Chang,
  • Jianshu Zhang,
  • Sharon L. Trevor,
  • Kristen S. Gibbons,
  • Stephen J. McPherson,
  • Jessica C. Kling,
  • Luregn J. Schlapbach,
  • Antje Blumenthal,
  • Lachlan J. M. Coin,
  • Sainath Raman,
  • Natalie Sharp,
  • Natalie Phillips,
  • Adam Irwin,
  • Ross Balch,
  • Amanda Harley,
  • Kerry Johnson,
  • Zoe Server,
  • Shane George,
  • Keith Grimwood,
  • Peter J. Snelling,
  • Arjun Chavan,
  • Eleanor Kitkatt,
  • Luke Lawton,
  • Allison Hempenstall,
  • Pelista Pilot,
  • Kristen S. Gibbons,
  • Renate Le Marsney,
  • Carolyn Pardo,
  • Jessica Kling,
  • Stephen J. McPherson,
  • Anna D. McDonald,
  • Seweryn Bialasiewicz,
  • Trang Pham,
  • Lachlan J. M. Coin

摘要

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.