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Related Concept Videos

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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing.

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|May 12, 2015
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Summary
This summary is machine-generated.

Sanger confirmation of next-generation sequencing (NGS) single-nucleotide variants (SNVs) is often unnecessary. However, Sanger sequencing may still be needed for insertion/deletion variants (indels) and quality assurance in NGS variant detection.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Clinical Diagnostics

Background:

  • Current clinical guidelines lack definitive recommendations for confirming next-generation sequencing (NGS) variants.
  • Sanger confirmation of NGS results can introduce inefficiencies, redundancy, and increased costs in laboratory practice.

Purpose of the Study:

  • To evaluate the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels).
  • To assess the necessity of NGS variant confirmation using Sanger sequencing or public databases.

Main Methods:

  • Utilized four NGS target-capture gene panels (117 genes, 568 Kbp) with 77 patient DNA samples.
  • Compared 1080 SNVs and 124 indels detected by NGS against Sanger confirmation and/or the 1000 Genomes Project (1000G) database.
  • Ensured depth of coverage exceeded 100× in >99.7% of target bases.

Main Results:

  • Achieved 100% concordance between NGS and Sanger for 919 recurrent variants.
  • Observed 97.1% concordance between NGS results and 1000G phase 1 data for 762 variants (736 SNVs, 26 indels).
  • Sanger sequencing and 1000G phase 3 data validated NGS accuracy for all discrepancies.

Conclusions:

  • Confirmatory Sanger sequencing for SNVs detected by high-quality capture-based NGS is redundant.
  • Sanger sequencing may be necessary for indel variant location refinement and for general NGS quality assurance.
  • Optimizing variant confirmation strategies can improve laboratory efficiency and reduce costs.