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Rare Event Detection Using Error-corrected DNA and RNA Sequencing
JoVE Journal
Genetics
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JoVE Journal Genetics
Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

DOI:
10.3791/57509-v

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10:36 min

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August 03, 2018

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, , ,
1Department of Pediatrics, Division of Hematology and Oncology,Washington University School of Medicine, 2Center for Genome Sciences and Systems Biology,Washington University School of Medicine

Chapters

  • 00:04Title
  • 00:33Incorporating i5 and i7 Adapters onto Error-corrected Sequencing Libraries
  • 03:36Quantifying the Libraries with QX200 Digital Droplet PCR (ddPCR)
  • 06:14Amplifying and Normalizing the Libraries for Sequencing
  • 07:37Results: Mutation-calling with Error-corrected Sequencing (ECS)
  • 09:14Conclusion

Summary

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Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5–2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Tags

Keywords: Rare Event DetectionError-corrected DNA SequencingError-corrected RNA SequencingClonal Single-nucleotide VariantsSmall IndelsUnique Molecular IdentifiersQ5 Master MixPCRMagnetic Bead CleanupIllumina ChemistryGene Panels

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