Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Detection of Central Nervous System Pathogens

Metagenomic next-generation sequencing (mNGS) has emerged as a powerful tool for unbiased pathogen detection and host transcriptional profiling in clinical and research settings. While its utility in diagnosing central nervous system (CNS) infections is increasingly recognized, cerebrospinal fluid (CSF) samples pose unique challenges due to low nucleic acid abundance and susceptibility to degradation. This study presents an optimized research-based protocol for Illumina sequencing platforms tailored to CSF mNGS, spanning sample handling, nucleic acid extraction, library preparation, sequencing, and bioinformatic analysis. Quality control approaches adaptable to both high and low-resource settings are also provided, including alternatives to capillary electrophoresis. This study demonstrates the protocol's robustness through two representative cohorts: a high-depth, NovaSeq-based workflow and a cost-conscious NextSeq-based workflow. Across these cohorts, pathogens were detected in over 50% of cases, underscoring the method's diagnostic potential even with resource-constrained adaptations. This protocol facilitates reproducible CSF mNGS, providing a foundation for diverse applications in neuroinfectious disease research and diagnostics.