Journal: Infection and Drug Resistance
Article Title: High-throughput and Efficient Assay for Central Nervous System Infection with Targeted Nanopore Sequencing Technology
doi: 10.2147/IDR.S540638
Figure Lengend Snippet: 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 abundance of each taxon at the species level (with –l S), using a read length of 300 bp (the shortest read length in the dataset) and a threshold of 10 reads (with –t 10) to filter out low-abundance species and reduce noise. The results of bioinformatics analysis were finally visualized by Pavian and GraphPad.
Article Snippet: The 25 μL multiplex PCR master mix contained 1× Q5 Hot-Start HiFi Buffer (New England Biolabs, Ipswich, MA, USA), 200 μM dNTPs, 0.2 μM each primer, 1× High GC enhancer, 0.5 U Q5 Hot-Start High-Fidelity DNA Polymerase (New England Biolabs), and 10 μL template DNA.
Techniques: Infection, Bacteria, Control, Amplification, Multiplex Assay, Nanopore Sequencing, Sequencing
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