Next-generation sequencing (NGS) analyses have revolutionized understanding of biological processes. In many basic science or clinical studies, substantive insights have been made by comparing the primary DNA sequences of genes in different groups of subjects. But as every researcher knows, no single application or technique tells the whole story. Often, it is the thoughtful combination of approaches that delivers the insight needed to move research forward.
Many labs specialize in sequencing for confirmation either as outsourced sites or core hospital sites. This secondary Sanger confirmation is especially important for clinical decision-making, where the validation of the mutation has real-world implications. Confirmation of a diagnosis or genetic testing in a person who believes they have a disposition toward a disease is essential. The current best practice in many labs is to confirm NGS with Sanger sequencing.
Application note: Using Sanger capillary electrophoresis sequencing to confirm variants discovered by next-generation sequencing (NGS)
A study found that 2% of the variants detected by NGS were not reproducible and required additional confirmation by Sanger sequencing. This application note discusses why confirmation is an important part of any analysis of NGS-based data and how Applied Biosystems reagents and systems facilitate the orthogonal verification of NGS results.
Sanger sequencing workflow
We offer a simple, fast, and complete workflow for verifying variants discovered by NGS systems. The workflow can be completed in less than one work day, from sample to answer. Applied Biosystems products are optimized to work together. NGS variants that are marked for verification can be directly imported into the Primer Designer Tool, where PCR primer pairs can be chosen and ordered for use in cycle sequencing. The resulting data are then aligned and compared in the Next-Generation Confirmation (NGC) tool in our Connect cloud-based platform.
Minor variant detection
Applied Biosystems Minor Variant Finder Software is new software developed for the detection and reporting of minor mutations by Sanger sequencing. For high-throughput labs using NGS technology, minor variants from NGS findings can be confirmed with Sanger sequencing quickly and cost-effectively. The innovative algorithm of Minor Variant Finder Software neutralizes the background noise using a control sample, and enables calling of minor variants at detection levels as low as 5%.
- Variant Venn diagram: shows the number of NGS variants listed in the .vcf file selected in Setup, and the number of unreviewed or accepted Sanger variants (variants rejected in the Variants screen are not shown). The intersection of the two circles indicates the number of variants that are identified as both NGS and Sanger variants.
- Annotated Variant: click to display NCBI information about the variant.
- Reference base at the variant location and alternate base identified by the NGS and Sanger analyses.
- Review Status: status from the Variants screen.
For Research Use Only. Not for use in diagnostic procedures.