Evaluation of amplicon-based nanopore sequencing for foot-and-mouth disease viruses in clinical and environmental samples

Figures

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  Fig 1. Independent amplification and barcoding of the S-fragment enhance sequencing depth across varying viral loads. (A) Schematic of the original and modified protocols. In the original protocol (left), genomes are amplified from Pools A and B and assigned a single barcode. In the modified protocol (right), the S-fragment is isolated from Pools A and B, and they are amplified in three distinct parts, with multiple barcodes assigned to each amplicon for nanopore sequencing. The red dashed box highlights the isolation of amplification and barcoding for the S-fragment. The genomic bar represents each pool, including the S-fragment (orange), Pool A (green), and Pool B (blue), in its respective region. (B) Comparison of whole-genome sequencing depth between the original and modified protocols using serial dilutions (Ct 24, 27, and 30) of three representative FMDV isolates: O/CJ/SKR/2023, O/BAN11/2023, and A/Gimpo/SKR/2018. Solid lines and shaded areas represent the mean depth and standard deviation, respectively, of independent triplicate experiments for the original (blue) and modified (orange) protocols. Whole-genome coverage plots display enhanced 5′-terminal S-fragment recovery under…

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  Fig 2. Nanopore sequencing success depends on sample type. (A) FMDV-positive sample distributions by sample type. Whole-genome (B) or VP1 coding region (C) recovery analyses: left panel, number of “Recovered” (orange) and “Not recovered” (gray) samples; right panel, proportional success rate for each sample type. P-values denote significant differences between sample types, as determined by Pearson’s χ2 test (P < 0.001).

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  Fig 3. Sequencing quality exhibits an inverse trend relative to viral loads across diverse sample types. (A) Relationship between Ct values and WGS coverage. The scatter plots present genome coverages relative to Ct values for all 129 positive samples tested, stratified by sample type. Coverage and Ct range were compared by a Pearson’s χ2 test (P < 0.001). (B) Relationship between Ct values and sequencing depth. The plots display the sequencing depth achieved for samples with varying Ct values. Differences in sequencing depth among Ct ranges were evaluated by one-way ANOVA (****P < 0.0001, ns, not significant). In all panels, the data points are colored by WGS success status (orange: complete genome, defined as 100% coverage at > 50× depth; gray: partial-genome recovery).

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  Fig 4. Amplicon amplification efficiency varies depending on genomic position but is consistent across the sample types. (A) Schematic of the FMDV genome and six overlapping amplicons. Amplicons are numbered 1–6 (5′–3′) according to their genomic positions. (B and C) Amplification success rates for amplicons 1–6 categorized by sample types (B) and Ct value ranges (C). (D) Overall amplification success rates based on genome position, summarizing the performance of each amplicon (1–6) across the 92 samples that failed to achieve complete WGS recovery.