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Updated: Aug 23, 2025
Sequencing of mRNA from Whole Blood using Nanopore Sequencing
Published on: June 3, 2019
Performance of Nanopore and Illumina Metagenomic Sequencing for Pathogen Detection and Transcriptome Analysis in
Kazuhiro Horiba1,2,3,4, Yuka Torii3, Yuta Aizawa5
1Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
Background:
Infantile central nervous system infections (CNSIs) can be life-threatening and cause severe sequelae. However, the causative microorganism remains unknown in >40% of patients with aseptic infections. This study aimed to analyze the metagenome for detection of pathogens and the transcriptome for host immune responses during infection in a single cerebrospinal fluid (CSF) sample using 2 different next-generation sequencing (NGS) platforms, Nanopore and Illumina.
Methods:
Twenty-eight CNSIs patients (<12 months) were enrolled, and 49 clinical samples (28 CSF and 21 blood) were collected. The DNA extracted from all 49 samples was sequenced using the Illumina sequencer for the detection of pathogens. Extracted RNA was obtained in sufficient quantities from 23 CSF samples and subjected to sequencing on both Nanopore and Illumina platforms. Human-derived reads subtracted during pathogen detection were used for host transcriptomic analysis from both Nanopore and Illumina sequencing.
Results:
RNA metagenomic sequencing using both sequencing platforms revealed putative viral pathogens in 10 cases. DNA sequencing using the Illumina sequencer detected 2 pathogens. The results of Nanopore and Illumina RNA sequencing were consistent; however, the mapping coverage and depth to the detected pathogen genome of Nanopore RNA sequencing were greater than those of Illumina. Host transcriptomic analysis of Nanopore sequencing revealed highly expressed genes related to the antiviral roles of innate immunity from pathogen-identified cases.
Conclusions:
The use of Nanopore RNA sequencing for metagenomic diagnostics of CSF samples should help to elucidate both pathogens and host immune responses of CNSI and could shed light on the pathogenesis of these infections.
Insights
Nanopore RNA sequencing effectively detects pathogens and analyzes host immune responses in infantile central nervous system infections (CNSIs). This advanced metagenomic diagnostics approach in cerebrospinal fluid (CSF) offers greater pathogen detection depth than Illumina.
Area of Science:
- Molecular Biology
- Infectious Diseases
- Genomics
Background:
- Infantile central nervous system infections (CNSIs) are critical, often with unknown causative agents in over 40% of aseptic cases.
- Accurate diagnosis is crucial for effective treatment and management of severe sequelae in affected infants.
- Current diagnostic methods may not fully identify all causative pathogens or elucidate host responses.
Purpose of the Study:
- To evaluate the utility of metagenomic and transcriptomic analysis using Nanopore and Illumina next-generation sequencing (NGS) platforms.
- To detect causative pathogens and analyze host immune responses in a single cerebrospinal fluid (CSF) sample from infants with CNSIs.
- To compare the performance of Nanopore and Illumina sequencing for metagenomic diagnostics.
Main Methods:
- Collected 49 samples (28 CSF, 21 blood) from 28 infants (<12 months) with CNSIs.
- Performed DNA sequencing on all samples using Illumina for pathogen detection.
- Conducted RNA sequencing on 23 CSF samples using both Nanopore and Illumina platforms for pathogen and host transcriptomic analysis.
Main Results:
- RNA metagenomic sequencing identified putative viral pathogens in 10 cases across both platforms.
- Illumina DNA sequencing detected 2 pathogens.
- Nanopore RNA sequencing demonstrated greater mapping coverage and depth for detected pathogen genomes compared to Illumina, with consistent results.
- Host transcriptomic analysis via Nanopore sequencing revealed highly expressed antiviral innate immunity genes in pathogen-identified cases.
Conclusions:
- Nanopore RNA sequencing is a valuable tool for metagenomic diagnostics of CSF samples in infantile CNSIs.
- This approach can simultaneously elucidate both pathogens and host immune responses, aiding in understanding infection pathogenesis.
- The findings support the use of Nanopore RNA sequencing for comprehensive diagnostics in pediatric neurological infections.
