Table 3.
Troubleshooting
| Steps | Problem | Possible reason | Possible solution |
|---|---|---|---|
| 5 | A260/280 is low | Proteinase K digestion is not sufficient for removing all the protein | Extract the DNA with Phenol-Chloroform twice |
| 9 | Average size of DNA smear is too large | Insufficient sonication or incompletely dissolved genomic DNA | Perform 1-2 more sonication cycles. |
| 36 | Very low DNA concentration (<3ng/μl) | Melting temperature (Tm) is not optimal for steps 11, 29 | Test the primer by gradient PCR and choose the right Tm |
| Wrong primer and/or dNTP concentrations for step 10 | Use correct concentrations for step 10 | ||
| Wrong pair of primers for steps 11, 29 | Check the sequences of the primers and make sure the nested primer corresponds with the bio primer | ||
| Bait DSB site is not cutting | Check amplified region to ensure target sequence is present; test another nearby target site | ||
| The bridge adapter is thawed and frozen too many times | Use fresh aliquot of bridge adapter | ||
| Beads were spun to bottom before ligation or nested PCR at steps 22, 29 | Do not spin the mixture before PCR | ||
| Operation error in some step | Re-do on-bead PCR or start over | ||
| Too high DNA concentration (>50ng/μl) | Unspecific priming | Design new primers | |
| Test background in an untreated (uncut bait DSB) library | |||
| 40 | Very low DNA concentration (<2ng/μl) | Blocking enzyme sites on the bait region or the I7-Blue primer | Change blocking enzyme |
| Operation error in some step | Re-do on-bead PCR or start over | ||
| 44 | Very short DNA smear tail | Too few PCR cycles | Increase the PCR cycles |
| Very long DNA smear tail | Too many PCR cycles | Reduce the input DNA amount for step 41 or decrease PCR cycles | |
| 49,51 | Pipeline does not execute | Incorrect metadata file | Check to make sure primer sequences match specified coordinates |
| 49 | No reads | Wrong barcode sequence | Check the sequences of the barcode and primer |
| Multiple samples with identical barcode and primer sequence at step 427 | Run identical barcode/primer samples on separate Miseq runs | ||
| Operation error for step 41 | Re-do step 41 | ||
| 51 | Very few junctions | Poor cutting at the bait DSB | Verify sufficient cutting at bait DSB |
| Primers are not annealing properly | Check amplified region to ensure primers do not overlap with a polymorphic site | ||
| Too high background | Cells are unhealthy or dying | Make sure the treatment doesn't cause too much DNA damages to the cells | |
| Many junctions in uncut-cell control | Repetitive sequence in bait region | Design a new bait DSB site |