Sanger Confirmation for NGS
The Sanger Method, it’s the gold standard for DNA sequencing—but how valuable is it in today’s lab? Sanger has one purpose – to sequence single strands of DNA with a high degree of certainty. It has been around since the 1970’s and though it is an old tool in the genomics tool kit, it is still relevant. Sanger can be used to validate results from NGS, confirm cloned inserts or mutated sequences, identify disease-causing genetic sequences, target small regions, and HLA typing. It is a cost-efficient way of sequencing single samples. Though every sample has a cost, it is far less expensive than running a high-throughput NGS sequencer for a single sequence. The current best practice in many labs is to confirm NGS sequencing with Sanger.
Sanger Sequencing for Clinical Labs
There has been debate in the scientific community about the continued need for Sanger sequencing. While some say NGS is enough to find genetic variants, a recent study in The Journal of Molecular Diagnostics found the chance for false-positives while testing for difficult-to-detect, disease associated variants was 1.3%, while controlling the bioinformatics pipeline to eliminate those false results would result in missing 2% of real mutations.
Adding the additional step of Sanger sequencing to workflows understandably adds turn-around time and expense to projects. However, there are many labs that specialize in sequencing for validation (either as outsourced sites or core hospital sites). This secondary Sanger confirmation is especially important for clinicians making a diagnosis or deciding on treatment options, where the validation of the mutation has real-world implications.
The extra day needed for confirmation is inconsequential when a life may hang in the balance. Confirmation of a diagnosis for an ill patient or genetic testing in a person who believes he/she has a disposition toward a disease is essential. For instance, a person who tested positive for an abnormal BRCA1, BRCA2 or PALB2 gene should have secondary confirmation before considering something as serious as a prophylactic mastectomy.
Sanger Sequencing in Biopharma Labs
Sanger is not the sequencing method traditionally associated with biopharmaceutical development. However, “low tech” Sanger sequencing has a home in the “high tech” world of biopharma R&D, and manufacturing. Labs can rely on both Sanger and NGS, using NGS to meet large-scale needs and Sanger for smaller projects.
There are times when NGS would not be the best use of a lab’s resources. For a biopharma, where testing and creating new large molecules is part of the R&D process, Sanger sequencing helps in sequence validation. Was the experiment a success? Did the team create the correct mutation and what was the result? Instead of taking the time and space in a NGS sequencer to answer these questions, an organization can increase efficiency by using Sanger for small validations and by reserving NGS sequencers for larger scale projects.
Sanger is necessary when moving from upstream bioprocess R&D to downstream production. The ability to trace the sequencing analysis of new modalities as the process moves to scale is vital. If there is a point in the process where results are no longer correct, the ability to check all data about the process, and analyze the samples/products from one step to another is important to pinpoint and correct problems.
Sanger and NGS Data in a Lab
When Sanger testing is done, often the amount of information (miniscule by NGS standards) is read and evaluated in a spreadsheet. The physical separation between the Sanger sequence information and the NGS sequence data opens the possibility for errors in the lab.
Sanger sequencing applications which allow sequence data to be stored on the same informatics platform which also houses NGS sequence data, reduces errors caused by transcription. The Sanger applications in Core’s App Marketplace can be coupled with an NGS workflow on the Platform for Science. This allows the information from the Sanger Sequencing to be directly compared with NGS results, leaving the data traceable, and reportable within the informatics platform.
Sanger lets you sequence one fragment at a time – which is a far more efficient use of time and energy if you only have a few samples to sequence. It is still considered the gold standard.
Chrisanne Wnek is a Genomics Application Specialist at Core Informatics. She holds a Master’s degree in Medical Laboratory Sciences from Quinnipiac University and is an ASCP certified technologist in molecular biology. She’s spent over 8 years working in the field of genetic research in Sanger, NGS and CLIA labs.