Table 1.
Dataset and troubleshooting
| Sample dataset | Production method | Platform | # CCS 3 | No. of aligned reads | # Total time, h | # SUM |
|---|---|---|---|---|---|---|
| AAV2-CB-EGFP (4,316 bases) | Triple-plasmid transfections | RSII | 19,494 | 19,369 | ∼0.8 | 83.8 |
| AAV2-CB-Luc (3,152 bases) | Sequel | 155,891 | 155,058 | ∼5.8 | 93.2 | |
| AAV8-hAAT-hLC (4,003 bases) | Sequel | 231,057 | 110,632 | ∼5.5 | 89.6 | |
| AAV8-TTR-hFVIII (5,123 bases) | Sequel | 55,300 | 52,470 | ∼2.6 | 53.6∗ | |
| AAV8-β-actin-hFVIII (4,993 bases) | Sequel | 154,403 | 152,774 | ∼6.0 | 83.0 | |
| AAV2-CMV-Luc (3,153 bases) | Sequel | 154,120 | 29,890 | ∼1.5 | 81.9 | |
| AAV2-hM4D.b (5,133 bases) | Baculovirus system | Sequel | 178,313 | 29,258 | ∼1.5 | 69.1∗ |
| AAV2-Luc.b (3,153 bases) | Sequel | 257,458 | 25,000 | ∼1.0 | 90.1 |
| Troubleshooting step | Problem | Possible reason | Solution |
|---|---|---|---|
| 2–1 | Inefficiency of DNase digestion | Insufficient incubation or low concentration | Increase the length of incubation or DNase concentration. |
| 2–3 | Low DNA extraction and purity | 1. Incomplete lysis of capsid 2. Residual phenol or ethanol |
1. The addition of Proteinase K is recommended in lysis buffer for complete lysis of AAV capsid 2. Further purification of DNA extraction by PCR purification kit |
| 3–1 | Inefficiency of DNA extension Poor quality of extension product |
1. High DNA amount (>1 μg) 2. High incubation temperature (>60°C) 3. Insufficient incubation time (<30 min) |
1. No more than 1 μg DNA/100 μL should be used in the reaction system 2. Incubation can go no higher than 60°C 3. Increase the length of incubation to 1 h |
| 3–3 | Inefficiency of ligation | 1. Blunt-end ligation 2. Insufficient incubation time for ligation |
1. Modify DNA end by tailing A 2. Apply sticky end adapters 3. Increase the length of incubation for ligation to at least 3 h or overnight at room temperature |
| 4 | Low amount or poor-quality DNA library product | 1. Vortex time is too long (>10) during AMPure bead purification, damaging the DNA 2. There are residual beads in the final library product 3. Bead pellet has been overdried before elution |
1. Quick flick or brief vortex to mix the bead solution and library sample 2. To completely remove residual beads in the final product after adding elution by repeating the following step 1–2 times: place the tube back in the magnetic bead rack until the solution clears, and then slowly transfer the supernatant into a new clean tube 3. To avoid overdrying and cracking the bead pellet, air dry for ∼2 min |
| 6–4 | <TMCA_Rearrange.sh>: discarding the invalid data using such VBA code is time-consuming | If processing a large dataset | Scripts could be optimized and parallelized using multiple CPUs |
| 6–5 | <TMCA_Rearrange.sh>: detection of main configurations are calculated with low ratio | The complexity of foreign sequencing integration exists, as well as secondary combinations | We strongly recommend using appropriate online searching analysis tools |
| 6–6 | <TMCA_Visualization.sh>: some defined configuration is detected with low abundance | The number of viral sequences detected in 1 sample is not exactly a real virus abundance, because of sequencing bias | Subsample varying length filtering is needed before format grouping |
# CCS_3: HiFi reads with an accuracy of >99% and with passes ≥3; # total time, h: for processing aligned reads, after Minimap2 alignment process, including BLAST and visualization; # SUM: the overall ratio of AAV genome molecules, including those with full, SBG, ICG, and GDM configurations, as well as their secondary derivative genomes, in the sequencing reads. SUMs of AAV-TTR and AAV-hM4D.b are low (∗), and further details can be found in the limitations section.