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. 

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NGS confirmation workflow


1 Isolate DNA
identify variants
2 Perform PCR
choose primers
3 Prepare for sequencing
prepare

Prepare for sequencing using BigDye Direct Cycle Sequencing Kit

4 Run sequencing
run sequence

Run sequencing using 3500xL Genetic Analyzer

5 Analyze data
analyze data

Analyze data using Minor Variant Finder Software

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.

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sanger sequencing

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

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.

Sanger Sequencing for Genome Finishing

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)

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.

Visualization of NGS confirmation data
  1. 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.
  2. Chromosome
  3. Annotated Variant: click to display NCBI information about the variant.
  4. Reference base at the variant location and alternate base identified by the NGS and Sanger analyses.
  5. Review Status: status from the Variants screen.

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

Visistat reference component

Support

New Capillary Electrophoresis Instruments Support Center
Find tips, troubleshooting help, and resources for support related to your capillary electrophoresis instruments.