Table 9 Troubleshooting table
Step | Problem | Possible reason | Solution |
|---|---|---|---|
20 | Low PCR yield | Poor vectorette adapter annealing or ligation | Anneal fresh vectorette adapters and repeat procedure |
20 | Low PCR yield | Low starting gDNA quality/quantity | Increase the initial amount of starting gDNA, or isolate fresh gDNA |
21 | Very high molecular weight smear | Vectorette-Primer concatemer amplification | Digest 2āĪ¼g of vectorette PCR amplicons with BstYI and running on a 1.5% agarose gel. An intense band around 50ābp indicates the presence of concatemers in the PCR product (see AdditionalĀ fileĀ 4: Figure S1B). Repeat procedure with fresh reagents in an amplification-free area. |
27 | Library yield too low to sequence | DNA lost during library preparation or size-selection | Restart library preparation with more sheared DNA (0.5-1āĪ¼g) |
28 | Uneven sequencing output distribution | Uneven library pooling | Performing qPCR on prepared libraries with KAPA Library Quantification Kit prior to pooling may result in a more balanced sequencing output. |
30 | High number of overlapping read pairs | Small library fragments | Add a Pippin prep selection after pooling (step 28) to remove fragments under 400ābp. |
TableĀ 7, step 5 | No L1 insertion band | Large/difficult L1 insertion | Use L1-specific 3ā PCR (see TableĀ 8) |