High-throughput and Efficient Assay for Central Nervous System Infection with Targeted Nanopore Sequencing Technology

Figures

• 

  Figure 1. Workflow of tNPS-based experimental and bioinformatics analysis for detecting pathogens of CNS infection. (A) Genomic DNA was first extracted from samples using boiling method. Second, the target regions of genes related to 17 high-priority in CNS infections (seven bacteria, one fungus, and nine viruses), the full-length gene regions of 16S rRNA and ITS, and three internal control plasmids were then amplified by multiplex PCR in a bacterial-fungal tube and a viral tube. Third, the amplicons were mixed and pooled for library preparation. Fourth, the library was subjected to nanopore sequencing to generate long-read sequence data for bioinformatics analysis. The turnaround time was approximately 8 h. (B) MinKNOW first collected real-time data, which were converted to FASTQ format (raw reads) by Guppy. Second, Q_score filtered the raw reads into clean reads with base quality values >9. Third, using Bowtie 2, the human reads were removed by matching human genome reference sequence (GRCH38.p14). Kraken 2 was followed to map these reads to the core_nt database for species classification, with the parameter “--minimum-hit-groups” set to 3 to improve accuracy. Bracken also estimated the…

• 

  Figure 2. Data qualities of reference strains and mock communities using tNPS. (A) Raw and clean reads from 12 reference strains, Zymo (Zymo1 and Zymo2 were replicates), SC-BF (SC-BF1 and SC-BF2 were replicates), and SC-V samples. (B) Scatter plot comparing the mean base quality scores (Q) between raw and clean reads from ONT R10.4.1 sequencing platform. Mean (Q) raw reads = 16.95, clean reads = 17.20 (Student’s t-test). ****P <0.0001.

• 

  Figure 3. Sankey plots of tNPS results from reference strains. (A–C) C. neoformans, N. meningitidis and EBV, were identified along with the three internal controls (S. ruber, T. koningii and CaMV), respectively. Sankey plots of Pavian showed the distribution of species taxonomic abundance at the levels of D, domain; P, phylum; G, genus; and S, species. Different colors represented distinct classifications, and the width of the flow indicated their relative abundance.

• 

  Figure 4. Sankey plots of tNPS results from mock communities. (A–C) With the respective data derived from assaying Zymo, SC-BF and SC-V, the three mock communities were identified along with the three internal controls (S. ruber, T. koningii and CaMV).

• 

  Figure 5. Microbial classification and reproducibility analysis of tNPS. (A) Heatmap of Bray–Curtis dissimilarities between Zymo replicates and SC-BF replicates. Color intensity represented relative compositional differences, with darker hues indicating higher divergence. (B and C) Microbial abundance ratios in Zymo (Zymo1 and Zymo2) and synthetic communities (SC-BF1, SC-BF2 and SC-V) at genus and species levels. Bar heights represented relative abundance ratios of microorganisms.

• 

  Figure 6. Sankey plots of tNPS results from clinical samples. (A–C) VZV, S. pneumoniae and negative result, were identified along with the three internal controls (S. ruber, T. koningii and CaMV), respectively.