Sanger sequencing is highly effective for testing small targeted genomic regions and for validating results from NGS. However, as with all sequencing technologies, there are limitations and needs for troubleshooting.
If the sample that you are sequencing does not produce the desired read quality, we recommend starting with three basic troubleshooting steps.
The primer that is optimized for your PCR reaction may not always work with the Sanger reaction. This is due to using a set annealing temperature. To ensure that the primer can work at its optimal level within the Sanger reaction, the primer must fit into certain ratios, between 18 to 24 base pairs with 45 to 55% GC content at annealing temperature between 55 and 60 degrees Celsius.
If you are encountering poor-quality data or failed sequencing reactions, we recommend checking for any possible contaminants within the sample.
Check that the elution buffer does not contain EDTA — any buffers such as TE — as this can hinder the sequencing reaction and cause it to fail.
Check 260/230 of the samples. Low 260/230 (<1.6) suggests the presence of organic contaminants that can impact sequencing quality.
If ethanol precipitation is used for DNA template isolation, we recommend doing thorough washes to eliminate any remnants of ethanol, as this can also affect sequencing quality.
If the DNA template contains GC-rich hairpins or secondary structures around the primer binding region, or within the sequencing region, the Sanger reaction will be negatively affected.
For particularly difficult DNA templates, in the US we offer an innovative protocol to tackle these challenges head-on, enhancing the robustness of the Sanger reaction. In the EU we offer a difficult template troubleshooting option during the sequencing process. Reach out to one of our experts to learn more.
US: dnaseq@azenta.com | EU: Sanger.europe@azenta.com | UK: Sanger.UK@azenta.com
In order to achieve optimal sequencing results, it’s important to understand Sanger boundaries; what’s required to optimize its accuracy, and when you may benefit from other cost-effective sequencing solutions.
Good primer design is critical for successful Sanger sequencing. Poor quality primers fail to bind to the template, which leads to poor sequencing results.
Optimal primer characteristics include:
Tip: While preparing and using primers, please remember that pmol/µl = µM.
When it comes to primer design, you’ve got options.
In addition to primer design, another limitation of Sanger sequencing is sample concentration and volume.
For a Sanger reaction, the total concentration is based on the total length of the DNA submitted and not the region that will be sequenced. This is to calculate and validate the appropriate number of template copies within the sample.
For best results, we require that the sample concentration is as close to the recommended guidelines as possible. The recommended concentration has been optimized to work efficiently with the primer concentration for the best yield and quality. A higher concentration can always be diluted; in cases where the concentration is too low, the sequencing efficiency is negatively impacted.
Learn more by reviewing our sample submission guidelines.
For every Sanger reaction with a particular primer, we deliver up to 1100 base pair read length from the primer binding site.
However, there are cases where a longer read length is needed. Additional primers further downstream are then used to deliver the expanded coverage, which potentially increases the overall cost.
For cases like these, we recommend reaching out to our technical support specialists to discuss the best options for optimizing cost and read length, such as Plasmid EZ (with ONT) which enables cost-effective, rapid, and high-throughput sequencing of whole plasmids. Our dedicated support team is here to enable your success.
Sanger sequencing offers valuable insights into targeted genomic regions and serves as a robust tool for validation but can come with troubleshooting challenges such as primer design, contaminants, and template sequencing issues. To address these challenges, In the US, we offer an innovative protocol to tackle these challenges head-on, enhancing the robustness of the Sanger reaction. In the EU we offer a difficult template troubleshooting option in the sequencing process. Our proprietary solution can help to streamline your workflow, saving you critical research time.
GENEWIZ Sanger Sequencing offers solutions to navigate limitations but also provides access to universal primers, a personalized primer selection tool, and expert technical support, ensuring your sequencing endeavors are optimized for success. Experience the efficiency of our Sanger sequencing service and empower your research with our comprehensive support.equencing needs. Experience the power of ONT within our service and propel your genomic research forward with confidence.