Targeted sequence capture of coxsackievirus A6 using nanopore sequencing directly from clinical specimens

Figures

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  Fig 1. Diagram of genome organization of enteroviruses.

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  Fig 2. Serial dilution of CVA6-positive clinical samples. (A) Ct values of eight (S1–S8) CVA6-positive clinical specimens. (B) Flowchart of serial dilution. *A serial 10-fold dilution series was prepared from each original clinical specimen. Briefly, 500 µL of the thoroughly vortexed original sample was aliquoted into a new microcentrifuge tube to prepare the 100 (undiluted) sample. To prepare the 10−1 dilution, 100 µL of this aliquot was transferred into 900 µL of phosphate-buffered saline (PBS) and mixed thoroughly. Subsequently, 100 µL of the resulting 10−1 dilution was transferred into a fresh tube containing 900 µL of PBS to yield the 10−2 dilution. This process generated three concentration levels (100, 10−1, and 10−2) for each of the eight CVA6-positive samples, resulting in a total of 24 libraries for sequencing analysis.

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  Fig 3. The concentrations of the pooled PCR products from the 24 samples.

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  Fig 4. The depth of genomic coverage for CVA6 with time, across concentrations, and among samples using PromethION flow cell. Panel A shows the trends of the average depth data of genomic coverage for all samples in 16 h. Panels B–I show the entire depth data of genomic coverage for the eight clinical specimens with three serial dilutions (low, 10−2; medium, 10−1; and high, 100). 5′ UTR, translation and replication control (1–743 nt); VP4, mediates viral uncoating (744–950 nt); VP2, maintains capsid stability (951–1,709 nt); VP3, assists cell attachment (1,710–2,441 nt); VP1, attachment and neutralizing antigen (2,442–3,335 nt); 2A, cleavage and translation hijack (3,336–3,779 nt); 2B, modulates membrane permeability (3,780–4,079 nt); 2C, RNA helicase and replication (4,080–5,072 nt); 3A, replication membrane scaffold (5,073–5,336 nt); 3B (VPg), replication primer (5,337–5,408 nt); 3C, polyprotein processing (5,409–5,957 nt); 3D (RdRp), RNA-dependent RNA polymerase (5,958–7,340 nt); and 3′ UTR, RNA stability and replication (7,341–7,411 nt).

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  Fig 5. The relevance between data volume, target reads ratio, genomic coverage, and Ct value of the CVA6 genome. Panel A shows the plot of Ct values fitted to the CVA6 data volume and the target reads ratio. Panel B shows the scatter plot of CVA6 data volume versus genomic coverage. The blue dotted line represents the lowest genomic coverage in this study.

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  Fig 6. Comparative analysis of CVA6 genomes obtained using Nanopore and Illumina sequencing techniques. (A) NJ tree of CVA6 strains of this study. (B) Pairwise analysis of CVA6 genome sequences using Nanopore and Illumina sequencing techniques. (C) Single nucleotide polymorphism of consensus sequences of both sequencing techniques. G2, Illumina sequencing; G3, Nanopore sequencing of the undiluted clinical samples. Scale bar, nucleotide substitutions/site.

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  Fig 7. Near full-length genome-based phylogenetic analysis. The neighbor-joining tree was built using genome sequences of 24 libraries (●) and 50 references (▲). Scale bar, nucleotide substitutions/site; L, low (10−2); M, medium (10−1); and H, high (100, undiluted).