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Cell Biology JoVE (Open Access) Citable · DOI

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

DOI: 10.3791/57509-v
What you'll learn
  • ✓ Implement error-corrected sequencing to reduce NGS platform error rates
  • ✓ Detect rare mutations at variant allele fractions below 0.01%
  • ✓ Quantify and normalize error-corrected DNA/RNA libraries for sequencing
Protocol

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.

Difficulty
advanced
Total time
~3-4 days (library preparation through sequencing run)

Steps

1
Incorporate i5 and i7 adapters onto libraries

Attach sequencing adapters to error-corrected DNA and RNA libraries to enable multiplexing and compatibility with NGS platforms. This step prepares libraries for downstream quantification and sequencing.

▶ 00:33
2
Quantify libraries using digital droplet PCR

Use QX200 ddPCR to accurately quantify adapter-ligated libraries and determine concentration before normalization. This provides precise molecule counting for downstream amplification steps.

▶ 03:36
3
Amplify and normalize libraries for sequencing

Perform PCR amplification and equalize library concentrations to ensure balanced representation across samples during NGS run. Normalized libraries are ready for error-corrected sequencing.

▶ 06:14
4
Perform mutation calling with error-corrected sequencing

Analyze sequencing results using error-correction algorithms to detect rare mutations at variant allele fractions as low as 0.0001, overcoming standard NGS error limitations.

▶ 07:37
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