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.
Sanger Sequencing Troubleshooting
If the sample that you are sequencing does not produce the desired read quality, we recommend starting with three basic troubleshooting steps.
1) Primer Design
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.
2) Contaminants
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.
3) Difficult Templates
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.
- Close to the primer binding region can lead to potential for no priming
- Mid or towards the end of the template or the sequencing region poses the risk of a failed or diminished signal when sequencing through the template
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
Sanger Sequencing Limitations
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.
1) Primer Design
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:
- Approximately 18-24 bases in length
- Melting temperature (Tm) between 50-60 degrees Celsius
- GC content at about 45-55%
- G or C base at the 3′ end
- 3′ end is complementary to your template
Tip: While preparing and using primers, please remember that pmol/µl = µM.
When it comes to primer design, you’ve got options.
- Universal primers can be obtained free of charge from Azenta Life Sciences here.
- Looking for a different primer? In your Azenta account, under ‘My Tools’, upload your template sequence to the Universal Primer Selection Tool to highlight the available primer binding sites. From there, select which primer works best for your sample.
- Request a synthesized primer directly within your Sanger sequencing order.
- The option to submit your own primer with your sample for sequencing is also available.
2) Sample Concentration and Volume
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.
3) Longer Read Lengths = Increased Costs
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.
Elevate your Sanger Sequencing with our Service Solutions
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.
About the Author
Annie Huang
Annie Huang is a Marketing Manager for Genomics content strategy at Azenta Life Sciences. She holds a BS in Microbiology and Molecular Genetics from Michigan State University and has worked in Product Marketing, Technical Sales, and Market Development across the life sciences industry prior to her current role. Today, she oversees content management and strategy for Next Generation Sequencing, Synthetic Biology (Oligo, Gene Synthesis, Plasmid Prep), and PCR + Sanger Sequencing portfolios for GENEWIZ Multiomics & Synthesis Solutions from Azenta Life Sciences.