Next-generation sequencing (NGS) analyses have revolutionized our 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.
For example, a recent study found that up to 2% of the variants detected by NGS were not reproducible by Sanger sequencing. Therefore, before any firm conclusions are drawn from an NGS study, potential variants identified by NGS should be confirmed by an orthogonal method.
NGS confirmation workflow
Isolate DNA using RecoverAll Total Nucleic Acid Isolation Kit
Perform PCR with primers designed for Sanger sequencing panels
Prepare for sequencing using BigDye Direct Cycle Sequencing Kit
Run sequencing using 3500xL Genetic Analyzer
We offer a complete workflow for verifying variants discovered by NGS systems. 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 with the BigDye™ Direct Cycle Sequencing Kit and the 3500 Genetic Analyzer. The resulting data (.vcf files from NGS and .ab1 trace files from Sanger) are then aligned and compared in the Next-Generation Confirmation (NGC) tool in Thermo Fisher Cloud.
Application note: Using Sanger capillary electrophoresis sequencing to confirm variants discovered by next-generation sequencing (NGS)
This application note presents information to help researchers using next-generation sequencing– based data to confirm their results. We show why confirmation is an important part of any analysis of NGS-based data. In addition, we show how the reagents and systems available under the Applied Biosystems brand portfolio facilitate the orthogonal verification of NGS results.
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%.
An NGS variants file (.vcf) from any NGS platform can be imported into Minor Variant Finder Software under the reference tab for confirmation reporting. The confirmation data can then be visualized in an alignment view and Venn diagram.
- 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.
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