Schematic of the mNGS assay workflow. (A) CSF is extracted after lysis by bead-beating and internal control addition to allow viral, bacterial, fungal, and parasite nucleic acid retrieval. Total nucleic acid extracts are enriched for pathogen DNA by removal of methylated DNA (DNA libraries) and treatment with DNase (RNA libraries). (B) Libraries are generated using the Nextera XT protocol and amplified using two rounds of PCR. Libraries are quantified, pooled, and loaded onto the sequencer. (C) Sequences are processed using SURPI+ software for alignment and classification. Reads are preprocessed by trimming of adapters and removal of low-quality/low-complexity sequences, followed by computational subtraction of human reads and taxonomic classification of remaining microbial reads to family, genus, or species. For viruses, reads are mapped to the closest matched genome to identify nonoverlapping regions; for bacteria, fungi, and parasites, a read per million (RPM) ratio (RPM-r) metric is calculated, defined as RPM-r = RPMsample/NTC. To aid in analysis, automated result summaries, heat maps of raw/normalized read counts, and coverage/pairwise identity plots are generated for use in review and clinical interpretation.
