Table 2. Rates of detectable chimeras in sequence data.
Average rates of detectable chimeras are shown for each experiment performed in this study. Significantly lower rates of chimera formation were observed for DePCR-amplified gDNA samples relative to TAS-amplified samples, across multiple annealing temperatures. No significant difference in chimera formation was observed with DePCR methodology with varying gDNA input levels. Significantly higher chimera formation was also observed with TAS relative to DePCR when individual primer variants (RPVs) were utilized.
| Experiment | PCR Method | Annealing Temp. (°C) | Input concentration (ng/reaction) | Chimera detection rate [Average (SD)] | ANOVA |
|---|---|---|---|---|---|
| Annealing temperature | TAS | 40 | 10 | 5.16% (0.37%) | 1.41E-09 |
| DePCR | 40 | 10 | 0.05% (0.03%) | ||
| TAS | 45 | 10 | 6.49% (0.29%) | 4.05E-11 | |
| DePCR | 45 | 10 | 0.10% (0.07%) | ||
| TAS | 50 | 10 | 6.53% (0.21%) | 2.02E-12 | |
| DePCR | 50 | 10 | 0.04% (0.02%) | ||
| TAS | 55 | 10 | 5.69% (0.39%) | 9.66E-10 | |
| DePCR | 55 | 10 | 0.05% (0.02%) | ||
| TAS | 60 | 10 | 5.46% (0.49%) | 7.56E-09 | |
| DePCR | 60 | 10 | 0.03% (0.02%) | ||
| Input gDNA concentration | DePCR | 50 | 20 | 0.05% (0.02%) | 5.20E-01 |
| DePCR | 50 | 10 | 0.03% (0.03%) | ||
| DePCR | 50 | 5 | 0.03% (0.01%) | ||
| DePCR | 50 | 2.5 | 0.02% (0.01%) | ||
| DePCR | 50 | 1.25 | 0.03% (0.03%) | ||
| Reverse primer variants | TAS | 50 | 10 | 11.98% (3.85%) | 0.00 |
| DePCR | 50 | 10 | 0.06% (0.08%) |
Notes.
- SD
- standard deviation