Fig. 1

Development of the metagenomic next-generation sequencing protocol. A Overview of the method comparison study. There are ten methods named Microbial DNA-ILL, Microbial DNA-NEB, Total DNA-ILL, Total DNA-NEB, Total NA-ILL, Total NA-NEB, Total RNA-ILL, Total RNA-NEB, WTA-ILL and WTA-NEB. Microbial DNA-ILL: firstly the supernatant of CSF sample was removed, the microbial DNA was extracted after selective lysis then library was constructed with Illumina kit. Microbial DNA-NEB: firstly the supernatant of CSF sample was removed, the microbial DNA was extracted after selective lysis then the library was constructed with NEB kit. Total DNA-ILL: After the whole CSF sample was treated by lysozyme, the total DNA was extracted and retained then the library was constructed with Illumina kit. Total DNA-NEB: After the whole CSF sample was treated by lysozyme, the total DNA was extracted and retained then the library was constructed with NEB kit. Total NA-ILL: After the whole CSF sample was treated by lysozyme, the total nucleic acid was extracted then the library was constructed with Illumina kit. Total NA-NEB: After the whole CSF sample was treated by lysozyme, the total nucleic acid was extracted then the library was constructed with NEB kit. Total RNA-ILL: After the whole CSF sample was treated by lysozyme, the total RNA was extracted and retained by digesting genomic DNA then the library was constructed with Illumina kit. Total RNA-NEB: After the whole CSF sample was treated by lysozyme, the total RNA was extracted and retained by digesting genomic DNA then the library was constructed with NEB kit. WTA-ILL: After the whole CSF sample was treated by lysozyme, the total RNA was obtained and amplified by WTA kit then the library was constructed with Illumina kit. WTA-NEB: After the whole CSF sample was treated by lysozyme, the total RNA was obtained and amplified by WTA kit then the library was constructed with NEB kit. Each sample (sample1-14) had ten subsamples according to the ten methods. NA, nucleid acid; gDNA, genomic DNA; WTA, whole-transcriptome amplification using REPLI-g WTA Single Cell Kit; RT-PCR, reverse transcription PCR.B. Overview of Bioinformatic analysis. The raw sequencing data was preprocessed to gain clean data. Kraken2 was used to perform microbial classification. The positive cutoff of virus and nonviral pathogens were described in “Analysis of Results” in “Method”