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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

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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.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
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Targeted DNA Methylation Analysis by Next-generation Sequencing
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A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency

Page B McKinzie1, Michelle E Bishop1

  • 1Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079.

Toxicological Sciences : an Official Journal of the Society of Toxicology
|October 18, 2019
PubMed
Summary

This study presents a new, cost-effective method for quantifying low mutant allele frequencies (≥10-4) using next-generation sequencing. The technique is quick, easy, and suitable for detecting early cancer biomarkers.

Keywords:
KRASsingle-strand consensustargeted sequencing

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Accurate quantification of low mutant allele frequencies (VAFs) is crucial for clinical and nonclinical applications.
  • Existing methods like duplex sequencing offer high sensitivity but are complex and expensive.
  • There is a need for a more accessible method to quantify VAFs at frequencies of 10-4 and above.

Purpose of the Study:

  • To develop and validate a simple, quick, and cost-effective method for quantifying VAFs ≥10-4 using next-generation sequencing.
  • To assess the reliability and reproducibility of the proposed method for genetic biomarker detection.

Main Methods:

  • Developed a PCR fragment-based method for VAF quantification.
  • Tested the method using Kras codon 12 VAFs ranging from 10-5 to 10-1 and native genomic DNA.
  • Evaluated quantitation variability, error rates at different read lengths, and adaptability for multiplexing.

Main Results:

  • The method demonstrated a proportional increase in observed VAF to input VAF from 10-4 to 100% mutant samples.
  • Quantitation variability was consistent across experimental replicates and sample preparations.
  • Read lengths up to 70 bases were found to be reliable for quantitation of VAFs ≥10-4.

Conclusions:

  • The developed method provides an easy, quick, and less expensive alternative for quantifying VAFs ≥10-4 compared to existing techniques.
  • This approach is adaptable for various gene targets, multiplexing, and scalable for robotic handling, aiding in early carcinogenic biomarker detection.