Next-Generation Sequencing Validation | Thermo Fisher Scientific

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.

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. We have interviewed three researchers who are successfully employing various capillary electrophoresis (CE) applications to fill in some of the gaps or ambiguities in their next-generation sequencing (NGS) data. Read "Case studies in complementary capillary electrophoresis and next-generation applications" to find more about the utility of combining NGS and CE data to gain a more complete picture.

Sanger Sequencing for Mutation Analysis

Sanger sequencing provides fast and accurate analysis of the mutations present at a frequency of 20% or greater in a sample. The combination of the new BigDye Direct Cycle Sequencing Kit and a 3500 Series Genetic Analyzer provide the most comprehensive and reliable sequencing data.

Learn more about BigDye Direct Cycle Sequencing kit

Fragment Analysis for Somatic Mutation Detection

Detection of somatic mutations in samples like formalin-fixed, paraffin-embedded (FFPE) tissues may require higher levels of sensitivity and specificity than can be achieved with Sanger sequencing approaches. Fragment analysis provides solutions for achieving the high levels of sensitivity and specificity needed for somatic mutation detection.

Learn about the SNaPshot Multiplex Kit

Learn about a new technique mixing Sanger sequencing and fragment analysis

Sanger Sequencing for Genome Finishing

Sanger sequencing can perform in many situations where next-generation sequencing is limited, such as sequencing through difficult homopolymeric regions. Use the 3500 Genetic Analyzer for gap closure and to fill in regions of sequences that cannot be identified by next-generation sequencing approaches.

Download "Resolving homopolymeric ambiguities using PCR-based fragment analysis” application note

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.

Download app note ›

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.

Learn more about each step of our recommended Sanger sequencing workflow ›

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%.

Visualization of NGS confirmation data

Click image to enlarge

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.
Chromosome
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.

Learn more or download the demo version of Minor Variant Finder Software ›