Sanger Sequencing Repeat Policy
Troubleshooting Terms
The total cost of the order is dependent on the number of reactions performed by our technologists in the initial run, regardless of the quality of data returned. Any repeated reactions are performed as a courtesy and may be selected for re-sequencing based upon the analyst’s judgement when reviewing the results of the initial run. There is no additional charge for repeated reactions.
The first 60-70 bps of a sequence may be noisy or unreliable, as that region is directly after the primer binding site. ACGT only guarantees up to 750 bps of usable data for plasmids. If you are having trouble with sections of the sequence outside of the range of 60-750 bps, you may need to use additional primers for accurate results.
General
In the event that all reactions using the same template or primer fail, those reactions may not be repeated. The specific template or primer will be detailed in the results summary.
Reactions producing data with double peaks, mixed PCR sequences, repetitive sequences, or homopolymers will not be repeated. These outcomes are typically not a result of sequencing reaction failure, but are caused by the sequence itself or the primer used.
Reactions producing data with clear evidence of high GC content, hairpins, or other secondary structures will not be repeated with our standard reaction protocol. If hard to sequence regions are observed and noted as such in the results summary, it is recommended to place a new order for those reactions, with either our Hairpin or High GC protocol selected. Additional charges may apply.
Low Cost Option (LCO)
Troubleshooting will be performed on a subset of reactions that do not meet our quality standards after the initial run. Depending on troubleshooting results, the remainder of the order may or may not be repeated. High intensity data will not be diluted and repeated, as samples should be premixed to the concentrations specified in our guidelines.
Standard
Troubleshooting will be performed on a subset of reactions that do not meet our quality standards after the initial run. Depending on troubleshooting results, the remainder of the order may or may not be repeated. PCR products may be diluted in the event that the results show evidence of high intensity data.
Premium and Direct Colony Service
In most cases, if the initial reaction does not produce usable data (i.e. bad or noisy data), ACGT will repeat the reaction, including dilutions of high intensity data for both PCR products and plasmids. For higher volume orders and orders where quality checks reveal issues with sample quality, troubleshooting reactions may be performed and based on the result, the remainder of the order may or may not be repeated.
Troubleshooting Common Sequencing Problems
Learn more about common causes of Sanger sequencing issues and how to overcome them in order to get good, clean data.
Characterized by low base confidence levels, usually lacking in any base-calling or having bases that do not match the expected sequence. Signal may be too low to accurately distinguish individual base pairs.
| Potential Cause | Possible Solution |
|---|---|
Poor DNA Quality | Confirm by checking samples via nanodrop and agarose gel. The A260/A280 value should be around 1.7-1.9. The A260/A230 value should be at around 2.0 – 2.2. Anything higher or lower than these values may indicate an issue with the purity of your DNA. Re-prepare DNA if contamination is suspected. If your purity ratios are not within the recommended ranges, you may need to add an additional wash step to your protocol, prior to elution. |
Incorrect Elution | We recommend using nuclease-free water to elute your DNA, instead of buffers as recommended by certain kits. Some buffers may contain sequencing inhibitors, such as EDTA. |
Template or Primer Concentration Too Low or Too High | Make sure your DNA concentration is consistent with our Sample Submission Guidelines for the service level chosen. If you are unable to check the concentration in your lab, submit your samples under the Premium service and we will perform QC checks prior to sequencing to ensure the correct amount of DNA is used. |
Lack of Primer Binding | Double check that the primer binding site is present, and that the primer is designed/selected properly. Sometimes, primers that are good for PCR are not the best for sequencing. Sequencing primers should be about 18-23 bases in length with a Tm of 56-62 °C. GC content should be between 40-60%. If you have chosen a primer that does not have a binding site along the template, you will need to choose or design a new one. Primers may also degrade over time. We do not recommend using primers that are older than 2 years. |
Seconday Structure Near Primer Binding Site | Our Hairpin protocol is designed to help sequence templates with tendencies to form hairpins or other secondary structures. |
Characterized by loss in signal quality or messy peaks prior to 750 bps or by background noise throughout the sequence. Part of the sequence may be clear and usable but may need to be closer examined to determine where quality becomes unreliable.
| Potential Cause | Possible Solution |
|---|---|
Poor DNA Quality | Confirm by checking samples via nanodrop and agarose gel. The A260/A280 value should be around 1.7-1.9. The A260/A230 value should be at around 2.0 – 2.2. Anything higher or lower than these values may indicate an issue with the purity of your DNA. Re-prepare DNA if contamination is suspected. If your purity ratios are not within the recommended ranges, you may need to add an additional wash step to your protocol, prior to elution. |
Incorrect Elution | We recommend using nuclease-free water to elute your DNA, instead of buffers as recommended by certain kits. Some buffers may contain sequencing inhibitors, such as EDTA. |
Template or Primer Concentration Too Low or Too High | Make sure your DNA concentration is consistent with our Sample Submission Guidelines for the service level chosen. If you are unable to check the concentration in your lab, submit your samples under the Premium service and we will perform QC checks prior to sequencing to ensure the correct amount of DNA is used. |
Dye Blobs | Dye blobs are very common in Sanger sequencing, and may most often occur within the first 100 bases of the sequence. These are often caused by the sequencing chemistry interacting with an unknown contaminate in the DNA or too high ethanol concentration used in cleanup. Resequencing can be requested if this occurs in a critical region. |
High GC Content | Our High GC protocol is designed to help sequence templates with GC-rich content (either across the template or concentrated in a particular region) |
Characterized by overlapping bases or visible presence of multiple PCR product ends.
| Potential Cause | Possible Solution |
|---|---|
Multiple Primer Binding Sites | Check sequence to see if there are multiple places that the primer could be binding to. You may need to redesign the primer to make it more specific to the region you are trying to read. You can try designing a primer from the opposite direction to bypass this issue as well. |
Contamination or Presence of Multiple PCR Products in the Sample | You may need to re-prepare the sample if there is potential contamination. Double check your gel picture to make sure that there is only one band present for the sample. If there are multiple bands, you may need to perform gel extraction for the desired product size. |
Repetitive Sequences and Homopolymeric Regions | With repetitive sequences, the polymerase can “slip”, creating double peaks after the poly-A, -G, -C, or -T region. The signal may drop dramatically or become completely unrecognizable after the repeat region. In this case, it may be best to design a primer in the opposite direction and align the sequences. You can also try using an anchored primer that will bind to the repetitive region (for example, our universal primers 21TA, 21TC, and 21TG) |
Characterized most often by an abrupt loss in signal, but may also appear as a gradual decline in signal or quality. Best determined by looking at the raw data.
| Potential Cause | Possible Solution |
|---|---|
High GC Content | Our High GC protocol is designed to help sequence templates with GC-rich content (either across the template or concentrated in a particular region) |
Hairpin / Secondary structures | Our Hairpin protocol is designed to help sequence templates with tendencies to form hairpins or other secondary structures. |
Characterized by electropherogram peaks that are much higher in signal, causing a compression of bases near the beginning of the sequence. Reads may clear up after the first 100-200 or so bps in plasmids and longer PCR products or may remain oversaturated throughout shorter fragments. Depending on the intensity, this can render results partially or completely unusable.
| Potential Cause | Possible Solution |
|---|---|
Template Concentration Too High | Too much sequencing product can oversaturate the capillaries of the sequencing instrument. Make sure your DNA concentration is consistent with our Sample Submission Guidelines for the service level chosen. If you are unable to check the concentration in your lab, submit your samples under the Premium service and we will perform QC checks prior to sequencing to ensure the correct amount of DNA is used. |