Performance of Nanopore and Illumina Metagenomic Sequencing for Pathogen Detection and Transcriptome Analysis in Infantile Central Nervous System Infections

Abstract

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 metagenome for detection of pathogens, and transcriptome for host immune responses during infection, in a single cerebrospinal fluid (CSF) sample using two 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 were sequenced using Illumina sequencer for the detection of pathogens. Extracted RNA were obtained in sufficient quantities from 23 CSF samples and these were 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 Illumina sequencer detected two 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 understand both pathogens and host immune responses of CNSI and could shed light on the pathogenesis of these infections.

Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses

“…Combining ONT with differential lysis methods, which remove host and non-encapsulated nucleic acids, can improve sensitivity (80,81) detection of bacteria and fungi, but this step may reduce sensitivity for certain microbes and reduce the ability to detect cell-free DNA and RNA, including viral nucleic acid (54,55). Furthermore, with increasing moves to combine host gene expression with microbial detection to improve infection-diagnosis rates (27,(50)(51)(52)(53), methods such as differential lysis, which deplete human nucleic acid may be less attractive.…”

“…Several studies highlight the power of host transcriptomics methods for distinguishing bacterial, viral, and non-infectious causes of illness (27,(50)(51)(52)(53), although none are being used diagnostically at present. When metagenomics does not identify any pathogens, such analysis could help distinguish between a non-infectious cause of disease and a lack of sensitivity of the metagenomics protocol.…”

“…Many metagenomic methods advocate depletion of host nucleic acid to improve sensitivity, especially where microbial abundance is low (48,49). However, depletion significatively reduces host transcriptomic information, which can, when combined with pathogen metagenomics, improve accuracy of diagnosis and provide important insights that inform patient management (27,(50)(51)(52)(53).…”

“…Many metagenomic methods advocate depletion of host nucleic acid to improve sensitivity, especially where microbial abundance is low (48,49). However, depletion significatively reduces host transcriptomic information, which can, when combined with pathogen metagenomics, improve accuracy of diagnosis and provide important insights that inform patient management (27,(50)(51)(52)(53). Human transcriptomic analysis can identify immune pathways upregulated in the host and can help distinguish between viral, bacterial, and non-infectious causes of disease, which is particularly important when no pathogens are detected through metagenomics (27,(50)(51)(52)(53).…”

Identification and Analysis of SARS-CoV-2 Mutation and Subtype using 2x tiled Primer Set with Oxford Nanopore Technologies Sequencing for Enhanced Variant Detection and Surveillance in Seoul, Korea

“…While the base quality of ONT is inferior to that of Illumina, multiplex PCR amplicon-based analysis can generate sufficient reads to compensate for some low base quality in both analysis methods [16, 17]. In addition, ONT analysis takes 8–24 h, whereas Illumina sequencing requires approximately 40 h or more [18].…”