Figure 3.
HA-QAP Is More Accurate than Classical Profiling Based on Relative Abundance in the Fungal Mock Experiments.
(A) Dose–response curves for linear correlation between read counts of spike-in plasmid (BI12-4) obtained by Illumina sequencing and amount of spike-in plasmid in the DNA samples, indicating that fungal reads of the spike-in plasmid in the sequencing data reflect the amount of spike-in plasmid in the initial DNA samples. The gray region indicates 95% CIs.
(B) Box plots representing the relative abundance of reads assigned to the three fungi in the same mock experiment with a gradient of spike-in levels from 0 to 8.0 × 105 copies per reaction. Wilcoxon rank-sum test showed no significant differences in fungal relative abundance between the control group without spike-in and groups with different spike-in levels (4.0 × 104, 8.0 × 104, 1.6 × 105, and 8.0 × 105 copies per reaction, P > 0.05).
(C and D) The HA-QAP method revealed the significant increase in fungal load (two-sided t-test, P < 0.05), which could not be detected by the classical method based on relative abundance. Box plots showing a comparison of fungal profiles in mock experiments between group 1 and group 2 samples using RAP (C) and HA-QAP (D). Quantitative abundance represents the copy-number ratio of fungal ITS relative to plant genome.
(E and F) The HA-QAP method more accurately detected the increase in Basidiomycota levels (two-sided t-test, P < 0.05) and revealed that the quantitative abundance of Ascomycota did not change (two-sided t-test, P > 0.05) when the levels of the other fungal isolates increased. Box plots showing a comparison of fungal profiles in mock experiments between groups 1 and 3 using RAP (E) and HA-QAP (F). Quantitative abundance represents the copy-number ratio of fungal ITS relative to plant genome. Notably, RAP showed a spurious reduction in Ascomycota levels (Asco-AF1 and Asco-AF105), but HA-QAP did not.
(G) Scatter plot showing the ratio of errors between HA-QAP and RAP. Dots on the line with fixed slope = 1 represent errors from HA-QAP equivalent to those from RAP. All dots fall below the line with slope = 1, demonstrating that the HA-QAP method presents more real data. Data are based on comparisons (group 1 versus group 2; group 1 versus group 3) at different spike-in levels. Three groups of mock experiments were designed; n = 4, 3, and 5 for groups 1, 2, and 3, respectively.
All data shown in (C) to (F) are from samples with 4.0 × 104 copies of spike-in per reaction. The trend was consistent when using other spike-in levels. Asco, Ascomycota; Basi, Basidiomycota. See also Supplemental Figures 4 and 5.