He Jingni, Ganesamoorthy Devika, Chang Jessie J-Y, Zhang Jianshu, Trevor Sharon L, Gibbons Kristen S, McPherson Stephen J, Kling Jessica C, Schlapbach Luregn J, Blumenthal Antje, Coin Lachlan J M
Department of Clinical Pathology, The University of Melbourne, Parkville, Australia.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
BMC Infect Dis. 2025 May 13;25(1):692. doi: 10.1186/s12879-025-11078-z.
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.
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.
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.
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.
全血RNA测序已越来越多地用于寻找疾病状态的转录组特征。这些研究传统上利用cDNA的短读长测序，遗漏了RNA表达的重要方面，如异构体丰度差异和聚腺苷酸（poly(A)）尾长变异。
我们使用牛津纳米孔技术测序对12例确诊的细菌和病毒败血症患者全血中提取的天然mRNA进行测序，并与匹配的Illumina短读长cDNA测序数据结果进行比较。此外，我们还探索了聚(A)尾长变异、新转录本鉴定和差异转录本使用情况。
Illumina cDNA测序和纳米孔RNA测序之间的基因计数数据相关性很大程度上取决于分析流程的选择；纳米孔数据使用NanoCount，Illumina数据使用Kallisto，在基因水平上平均皮尔逊相关性最高，为0.927，在转录本异构体水平上为0.736。我们在细菌和病毒感染之间鉴定出2个具有差异多聚腺苷酸化的基因、9个具有差异表达的基因和4个具有差异转录本使用情况的基因。对聚(A)尾长的基因本体基因集富集分析显示，参与信号传导的基因的mRNA中长尾巴富集以及氧化还原酶分子功能中短尾巴富集。此外，我们检测到240种非人为的新转录本异构体。
纳米孔RNA测序和Illumina cDNA基因计数高度相关，表明这两个平台都适用于基因计数生物标志物的发现和验证。纳米孔直接RNA测序通过揭示额外的转录后和共转录生物标志物，如聚(A)尾长变异和转录本异构体使用情况，提供了额外的优势。
