Troubleshooting:
There are so many variables that contribute to a successful sequencing reaction, therefore there are countless different outcomes. We do our best to try to troubleshoot problem sequences and give you our best guess as to what might have gone wrong based on what we see and the limited amount of information that we have about your DNA. Here are just a few of the problems that we commonly see and their possible causes: (if you have specific questions regarding your sequence data, please contact us and we will do our best to help in any way that we can).
| Result | Possible Causes | Possible Solutions |
|---|---|---|
| Weak or noisy sequence (a high degree of background noise that interferes with data) |
Low DNA [ ], dirty DNA, innefficient primer annealing, difficult DNA content (i.e.homopolymer, G/C rich, dinucleotide repeat region) |
Increase [ ], re-prep DNA, select or design a new primer, ask that we increase the number of sequencing cycles |
| Failed sequence (no sequence data at all) | Low DNA [ ], innefficient primer annealing, missing primer site, contaminant in DNA prep (i.e. TE or salts), PCR primer did not work for sequencing | Increase [ ], ask that we increase the number of sequencing cycles, select or design a new primer, EtOH precipitation to remove contaminant or re-prep DNA |
| Mixed sequence (two or more sequences superimposed on top of each other) | More than one clone (clean plasmid sequence followed by mixed sequence where clone begins), multiple primer annealing sites, more than one PCR product | pick a new colony and start over, select or design a new primer, run PCR product out on a gel and gel purify and/or sequence with nested primers |
| "Top-heavy" sequence (sequence is very strong in the beginning and falls off early | DNA or primer concentration to high or too low which throws the balance of the sequencing reaction off, difficult DNA content | Dilute or concentrate and re-sequence, request that we run more sequencing cycles and/or add betaine to sequencing reaction |
Definition of terms we commonly use:
1. "Top-heavy" means that the sequence is very strong in the beginning and then tapers off to nothing, usually falling off early. This is typically because of an unbalanced reaction or difficult DNA content.
2. "Unbalanced reaction" is when the ratio of DNA to primer is off in either direction (too high or too low). This throws the balance of the cycle sequencing reaction off. The reaction mixture has just the optimal amount of ddNTP's so if there is too much or too little DNA or primer, the ddNTP's are used up in the beginning of the reaction and there are none leftover for the longer extension products.
3. "Stutter" is when the peaks sort of begin to overlap each other in the electropherogram. Typically, either the downstream peak shows a stutter under the upstream peak or visa-versa and can cause missed basecalls. This usually occurs when the primer does not anneal well or the polymerase slips after a homopolymer region.
4. "Secondary structure" occurs when the template folds back up on itself so the polymerase cannot extend past the region and the sequence ends abruptly. This usually occurs in G/C rich regions.
5. "Sequence reload (or re-injection)" is when we re-inject an exitsing sequence from its original well. We typically do this when the sequence has lost peak resolution early or has a noise spike that obscures the data. Sometimes, a sequence will inject better the second time.
6. "Sequence re-run" is different from a reload since we start over from scratch with a re-run. In other words, we set up the reaction all over again and re-sequence it. If your sequence fails due to an error on our part, we will automatically re-run the sequence free of charge. However, if you ask us to re-run a sequence and the results are the same as the first time, then we will have to charge you for the additional reaction. If the results are different from the first time, then we will not charge you.