Next-generation sequencing 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. 

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.

Click on the icons below to learn more about each step of the workflow.


identify variants

Select variants identified by next-generation sequencing

choose primers

Choose primers through the Primer Designer Tool

prepare

Simplify your sequencing workflow with Big Dye Direct

run sequence

Confirm with Sanger Sequencing and resequence using the 3500 Genetic Analyzer

analyze data

Compare Sanger Sequencing data with NGS data in NGC module

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.