Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts

Abstract

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

Fig 1. Scatter and violin-strip plots of the DNA concentrations at T₁.

Scatter plot of Qubit−DeNovix (Panel A), Qubit−NanoDrop (Panel B) and DeNovix−NanoDrop (Panel C). The multiple R-squared (R²), Spearman correlation (R_S), and corresponding p-values are provided. The line of equality is presented as a diagonal line and the dashed line corresponds to the linear regression model with formula as indicated. Violin-strip plots of the DNA concentrations at T₁ by DeNovix, NanoDrop, and Qubit (Panel D). Statistically significant differences (Wilcoxon test, p<0.001) are marked with **. Dav: DeNovix mean DNA concentrations, NDav: NanoDrop mean DNA concentrations, Qav: Qubit mean DNA concentrations,T₁: prior to freeze storing.

Fig 2. Scatter and violin-strip plots of the DNA concentrations at T₂.

Scatter plot of Qubit−DeNovix (Panel A), Qubit−NanoDrop (Panel B) and DeNovix−NanoDrop (Panel C). The multiple R-squared (R²), Spearman correlation (R_S), and corresponding p-values are provided. The line of equality is presented as a diagonal line and the dashed line corresponds to the linear regression model with formula as indicated. Violin-strip plots of the DNA concentrations at T₂ by DeNovix, NanoDrop, and Qubit (Panel D). Statistically significant differences (Wilcoxon test, p<0.001) are marked with **. Dav: DeNovix mean DNA concentrations, NDav: NanoDrop mean DNA concentrations, Qav: Qubit mean DNA concentrations, T₂: after freeze storing.

Fig 3. Scatter plot of the ratio of mean DNA concentrations measured by DeNovix and Qubit in function of the DeNovix A_260/280 purity ratio measured at T₁.

The plot area between 1.7 and 2.0 on the x-axis is highlighted and depicts the interval where DNA is regarded as pure. A more detailed view of this area is shown as an inset plot.

Fig 4. Violin-strip plots of the A_260/280 and A_260/230 ratio.

The A_260/280 and A_260/230 ratio for each timepoint and quantification platform is depicted in Panel A and Panel B, respectively. For each ratio, the proposed reference interval is indicated where contamination is minimal (A_260/280: [1.7,2.0], A_260/230: [2.0,2.2]). Statistically significant differences (Wilcoxon test, p<0.001) are marked with **. D280av: DeNovix mean values A_260/280, ND280av: NanoDrop mean values A_260/280. D230av: DeNovix mean values A_260/230, ND230av: NanoDrop mean values A_260/230. T₁: prior to freeze storing, T₂: after freeze storing.

Fig 5

Scatter plots of the A_260/280 and A_260/230 ratio for T₁ (Panel A and B) and T₂ (Panel C and D) between DeNovix and NanoDrop. The multiple R-squared (R²), Spearman correlation (R_S), and corresponding p-values are provided. The line of equality is presented as a diagonal line. The dashed line corresponds to the linear regression model with formula as indicated. For each ratio, the proposed reference interval is indicated where DNA is regarded as pure (A_260/280: [1.7,2.0], A_260/230: [2.0,2.2]). T₁, prior to freeze storing; T₂, after freeze storing.