542. Nanopore and Illumina sequencing for pathogen metagenomics and host transcriptomics of cerebrospinal fluid in infantile central nervous system infections
Authors: Horiba Kazuhiro , Torii Yuka , Yamaguchi Makoto , Haruta Kazunori , Okumura Toshihiko , Suzuki Takako , Kawano Yoshihiko , Kawada Jun-ichi , Hara Shinya , Giske Christian , Ogi Tomoo , Ito Yoshinori 
Published: December 2022

Background

Infantile central nervous system infections (CNSIs) can be life-threatening and severe sequelae can be observed in encephalitis and bacterial meningitis. The causative microorganism is unknown in &gt; 40% of patients with aseptic infections. This study aimed to analyze metagenome for detection of pathogen, and transcriptome for host reaction of infection in a single cerebrospinal fluid (CSF) sample using two different next-generation sequencing (NGS) platforms, Illumina and Nanopore.

Methods

Twenty-eight CNSIs patients (&lt; 12 months), treated between June 2012 and April 2020, were enrolled. A total of 49 clinical samples (28 CSF and 21 blood) from 28 patients were collected. Extracted RNA, which was obtained from 23 CSF in sufficient quantities, was sequenced using both Nanopore and Illumina platforms to compare their performances in the detection of pathogens. Human-derived reads subtracted during pathogen detection were used for host transcriptomic analysis from both Nanopore and Illumina sequencing. All the extracted DNA from 28 CSF and 21 blood, was sequenced using Illumina sequencer for the detection of pathogens. Data analysis was performed on the in-house PATHDET pipeline.

A flowchart for pathogen detection and transcriptome analysis

Pathogen detection was performed on the in-house PATHDET pipeline and transcriptome analysis was performed by using the secondary outputs.

Results

RNA sequencing of CSF samples in 23 cases revealed potential viral pathogens in 10 cases: coxsackievirus B5 (4 cases), coxsackievirus B4 (3 cases), coxsackievirus B2 (1 case), echovirus E7 (1 case), and human parechovirus 3 (1 case). DNA sequencing was performed in 28 cases using Illumina sequencing, and Proteus mirabilis (1 case, consistent with the culture test) and human parvovirus B19 (1 case) were detected. The results of Nanopore sequencing and Illumina sequencing were consistent. However, the mapping coverage and depth to the detected pathogen genome of Nanopore sequencing was superior to that of Illumina sequencing. MX1, ISG15, and OAS1 were differentially expressed genes in patients with identified pathogens via metagenomic NGS, both the Nanopore and Illumina sequencing, and were associated with antiviral roles in innate immunity.

A pie chart for patients with central nervous system infections (CNSIs)

Results of next-generation sequencing (NGS) and the detected pathogen candidates using DNA/RNA workflows.

Performance of Nanopore and Illumina sequencing for pathogen genome mapping

The sequencing output, which is 100,000 reads randomly extracted from original sequencing reads, was mapped to the pathogen reference genome to determine (A) mapping coverage and (B) mapping depth.

Volcano plot of all genes analyzed in Nanopore and Illumina sequencing

Volcano plots showing differentially expressed genes in the cerebrospinal fluid between identified-pathogen and unidentified-pathogen samples from (A) Nanopore sequencing and (B) Illumina RNA sequencing.

Conclusion

The use of Nanopore sequencing for metagenomic diagnostics of CSF samples should help to understand both pathogens and host immune responses of CNSI and could shed light on the pathogenesis of these infections.

Disclosures

All Authors: No reported disclosures.