My Sanger sequencing failed, now what?

Rerun Policy

A sequencing run generally fails due to poor sample quality or inadequate primers. Occasionally, it can also fail due to a machine or human error on our end. To cover these very rare cases, we established a re-run policy.

Failure rerun

All samples that fail (meaning there is no readable sequence) will be rerun automatically at no additional charge using the original samples. For larger failed orders, a few samples will be run, and if they work we will rerun the rest.

User-requested rerun

If you have a sample that you would like us to rerun, you need to place a new order. If the sample(s) runs better than the original, there will be no charge for the rerun. If the sample(s) runs the same or worse than the original sequencing, you will be billed for the order.

To place a re-run order, log into your account and place an order like normal. On the order form, check the box next to “Request a rerun” and type the original order number into the adjacent field.

For sequencing re-run, we will use the DNA sample we already have. The chemistry used for the rerun must also be the same as the original sequencing to qualify as a user rerun.

Poor sequencing results: common reasons

Insufficient DNA

How did you quantify your samples? Did you use a NanoDrop after purifying with the Thermo Fisher ExoSAP-IT kit? It generally yields unreliable results. If you used the NanoDrop, did you check on Qubit? There instruments are available at the Genomics Facility.

Elution

Did you elute your samples in water, and not TE, as recommended? Is there any ethanol carried over? Is there any EDTA in the buffer? The presence of ethanol or EDTA in the sample will make the sequencing fail.

Contamination with other nucleic acids

• If you tried to sequence a PCR product, it is possible that your purification did not remove properly all the PCR primer carryover and excess dNTPs. We recommend purifying PCR products with a commercial product available from Qiagen, Promega, or Thermo Fisher.
• The presence of leftover primers or dNTP that were not properly removed during the purification step will also induce a biased reading of the concentration of the target DNA on the NanoDrop. This will negatively affect the preparation of primer-template mix for Big Dye reaction, eventually leading to poor sequencing results.
• It is also possible that your PCR product is contaminate with other amplification products. In this case, a gel purification may be necessary. We recommend that you run your sample out on a gel and cut out and purify the band of interest.
• Even gel purified PCR products may contain more than one product. If you think you may have more than one PCR product in your samples, using internal primers instead of the PCR primers may be a solution. Using an internal primers specific to the desired product result in less interference from secondary sequences.

GC-rich, secondary structure and hairpin regions

To prevent common sequencing problems, our standard protocol uses the AmpliTaq FS enzyme, which offers improved through difficult templates, such as those with high G+C content, homopolymer regions, and secondary structure. We also add 5% betaine to every sequencing reaction, which helps eliminate secondary structure.

If you are having trouble with a high GC template and our standard reaction cannot solve the problem, we can run the reaction with a different chemistry (the dGTP Kit). This is not a rerun, but will be considered a new reaction and additional charges will apply.

If you are having trouble with a hairpin template and our standard reaction is not sufficient to overcome the secondary structure, we can run the reaction with a hairpin protocol that works about a third of the time.

If you would like us to run your samples using dGTP or as a hairpin, please specify it in the comments section of the order, both cost an extra $5 per reaction.

“Please use dGTP kit” or “Please use hairpin protocol”

Note that Secondary structure is the hardest problem to overcome, and often the solution is to sequence from the other end.

Primer Tm

We provide recommendations for the melting temperature of primers in the document: “Primer Design for Sequencing.pdf”

Please check the Tm of your primers vs. our recommendations.

Too small PCR product

If your PCR product is too small, your target DNA will be been removed with the cleanup phase. We recommend that you design PCR products to be at least 200bp.