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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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Sanger Sequencing01:57

Sanger Sequencing

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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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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Related Experiment Video

Updated: Jul 16, 2025

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
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Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

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Next-generation sequencing methodologies to detect low-frequency mutations: "Catch me if you can".

Vijay Menon1, Douglas E Brash2

  • 1Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT 06520-8040, USA.

Mutation Research. Reviews in Mutation Research
|September 16, 2023
PubMed
Summary

Detecting rare DNA mutations is crucial for health but challenging. New ultrasensitive sequencing methods significantly improve the detection of ultralow-frequency mutations, revealing that common methods often yield spurious results.

Keywords:
Duplex sequencingLow-frequency mutationsNext-generation sequencingRare variantsVariant allele frequency

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Last Updated: Jul 16, 2025

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Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues
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Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues

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Area of Science:

  • Genomics and Molecular Biology
  • Bioinformatics and Computational Biology
  • Clinical Diagnostics and Prognostics

Background:

  • Mutations, or changes in DNA, occur at varying frequencies in tissues, posing challenges for clinical applications due to their rarity.
  • Standard Next-Generation Sequencing (NGS) has limitations in detecting ultralow-frequency mutations (VAFs < 0.5%), hindering accurate diagnosis and research.
  • Inconsistent terminology and methodologies complicate the reliable characterization of mutation frequencies across different research groups.

Purpose of the Study:

  • To define terms and characterize the challenges in measuring ultralow-frequency mutations.
  • To review and describe recent innovations in sequencing methodologies designed to enhance the detection of rare DNA variants.
  • To highlight the practical applications and implications of these advanced sequencing techniques.

Main Methods:

  • Review and categorization of advanced sequencing techniques, including single-strand, tandem-strand, and ultrasensitive parent-strand consensus sequence methods.
  • Detailed description of methods like Safe-SeqS, SiMSen-Seq, o2n-Seq, SMM-Seq, DuplexSeq, PacBio HiFi, and others.
  • Discussion of approaches that quantify variant allele frequency (VAF) down to 10-5 and mutation frequency (MF) down to 10-7 per nucleotide, or even lower by analyzing large genomic regions.

Main Results:

  • Ultrasensitive sequencing methods enable quantification of VAF down to 10-5 and MF down to 10-7 per nucleotide.
  • Some methods can quantify MF below 10-9 per nucleotide or <15 errors per haploid genome by analyzing extensive non-repeated sites.
  • Ultrasensitive techniques demonstrate that many mutations previously observed at 0.5-1% VAF are spurious without these advanced methods.

Conclusions:

  • Recent innovations in sequencing technologies significantly improve the ability to detect and quantify ultralow-frequency mutations.
  • These ultrasensitive methods are essential for accurate clinical diagnosis, prognosis, toxicology, and identifying new disease causes.
  • Distinguishing between clonal expansion and independent mutations requires clear reporting of mutation frequency metrics (MFminI vs. MFmaxI).