Table 1.
BLASTn read assignments and qPCR results for two independently produced and sequenced rAAV samples (sample 1 and 2).
| A nanopore BLAST bins as percent of total hits | ||||
|---|---|---|---|---|
| Run 1 (sample 1) | Run 2 (sample 2) | |||
| Group/threshold | >500 nt | >1000 nt | >500 nt | >1000 nt |
| rAAV genome | 97.00% | 97.34% | 97.91% | 97.96% |
| pITR | 1.11% | 1.29% | 0.97% | 1.25% |
| pRepCap | 0.47% | 0.49% | 0.23% | 0.27% |
| pHelper | 0.25% | 0.24% | 0.17% | 0.17% |
| hg38 | 1.18% | 0.65% | 0.72% | 0.35% |
| B qPCR (and insilico fragmentation) results as percent of total measurable with 95% confidence interval | ||||
| Primer | Sample 1 | Sample 2 | (in silico) | |
| Bla | 2.0 ± 0.3% | 2.9 ± 0.4% | (1.79%) | |
| Rep | 0.22 ± 0.04% | 0.24 ± 0.04% | (0.13%) | |
| E4 | 0.062 ± 0.009% | 0.08 ± 0.01% | (0.10%) | |
A: Total contamination levels in both samples are independent of the read-quality thresholds tested here, however the individual share of contaminations shifts towards higher amounts of human genomic sequences for the lower threshold. B: qPCR results lay in comparable ranges to the sequencing results, although a larger discrepancy is seen for the second sample in terms of bla and for rep gene sequences in general. The in silico read fragmentation and binning to qPCR targets was performed for reads from run 2.