Table 4.
Calculation of the melting point confidence interval for each variant group based on real-time RT-PCR HRM curve analysis using LR3.HRM4 or LR3.HRM6 primer pairs a
|
Variant group |
2.5
th
percentile
b
|
97.5
th
percentile
c
|
Interquartile range (IQR) (75%-25%)
d
|
Number of outliers (>±1.5xIQR) |
Shapiro- Wilk test of normality (p)
e
|
Melting point interval without overlaps |
||
|---|---|---|---|---|---|---|---|---|
| Confidence (%) | ||||||||
|
LR3.HRM4 |
|
|
|
|
|
|
|
|
| Group I |
83.22 |
84.08 |
0.43 |
0.00 |
0.103e |
83.20 |
83.70 |
67.74 |
| Group II |
83.22 |
84.18 |
0.45 |
0.00 |
0.000 |
83.15 |
84.56 |
100 |
| Group III |
84.91 |
85.65 |
0.13 |
6.00 |
0.000 |
84.57 |
85.64 |
95.89 |
| Group VI |
85.35 |
86.28 |
0.15 |
12.00 |
0.000 |
85.65 |
86.37 |
92.96 |
|
LR3.HRM6 |
|
|
|
|
|
|
|
|
| Group I |
84.79 |
85.39 |
0.09 |
7.00 |
0.002 |
84.78 |
85.69 |
100 |
| Group II | 86.01 | 86.78 | 0.42 | 0.00 | 0.000 | 85.70 | 86.90 | 100 |
aThe data generated for each variant group was tested for normality in order to calculate the largest interval with the highest confidence without overlaps between variant groups. These intervals are indicated in bold. Intervals where all data points (100% confidence) fell within the maximum range, the limits were adjusted to the 2.5th to 97.5th percentile to incorporate a margin of error to ensure accurate classification.
b2.5th percentile is the melting point temperature where 2.5% of data points is less than or equal to that temperature.
c97.5th percentile is the melting point temperature where 2.5% of data points is greater than or equal to that temperature.
dInterquartile range is the interval where the middle 50% of melting point temperatures can be expected.
eAssume a normal distribution if p > 0.05, meaning approximately 95% of melting point temperatures of the variant group will be within ±1.96 standard deviations of the mean.