A universal primer-independent next-generation sequencing approach for investigations of norovirus outbreaks and novel variants

Figures

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  Figure 1. Relationship between the number of NoV RNA copies used as input and the obtained number of NoV reads before and after poly(A) capture NoV input was quantified with real time PCR and GGI and GGI standards and the total number of NoV genome copies used as input was calculated as NoV RNA copies (log10) and shown on the X-axis. The number of NoV reads obtained per million reads is shown on the Y axis (log10 scale). For each sample, the obtained NoV reads per million reads are shown both with poly(A) capture (filled squares) and without poly(A) capture (filled circles).

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  Figure 2. Relationship between Ct value (x axis) and the number of NoV reads per million reads (y axis, Log10). A robust regression analysis was performed in Prism (Robust Sum of Squares: 36.72).

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  Figure 3. Relationship between the log10 number of NoV reads (X axis) and the percentage of full-genome coverage (Y axis: solid line) and the average read depth (Y axis: dotted line).

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  Figure 4. Genome coverage and phylogenetic comparison. Genome coverages are shown on the left side of the figure for the following outbreaks: Ob-1 (a), Ob-2 (c), Ob-3 (e), Ob-5 (g), Ob-6 (i), Ob-7 (l) and Ob-8 (m and n). The range of reads in the coverage plots are shown to the left of each coverage plot and the position on the used reference sequence is shown on the top. Phylogenetic trees of the consensus sequences are shown on the right side of the figure for the following outbreaks: from Ob-1 (b), Ob-2 (d), Ob-3 (f), Ob-5 (h), Ob-6 (j), Ob-7 (l) and Ob-8 (o) and relevant reference sequences.

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  Figure 5. Read mappings for the recombinant NoV strain. (a) Mapping of reads to the reference sequence KM036380 (GII.P16_GII.13), (b) Mapping of reads to the reference sequence JX459908 (GII.Pe_GII.4_Sydney_2012), (c) Analysis of reads spanning the recombination junction region. This was performed by constructing an in silico reference sequence composed of pos. 4880 to 5051 of KM036380 joined with pos. 5068 to 5239 of JX459908 (the junction is marked with a “J”); reads were subsequently mapped to this reference sequence to identify junction-spanning reads.