Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

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.
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
PCR01:32

PCR

Overview
Sanger Sequencing01:57

Sanger Sequencing

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...
RNA-seq03:21

RNA-seq

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 microarray-based...
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer.

Communications medicine·2026
Same author

Enspyre: a novel enrichment technology for selected DNA variants using pyrophosphorolysis.

Nucleic acids research·2025
Same author

Development of a Machine Learning Model for Aspyre Lung Blood: A New Assay for Rapid Detection of Actionable Variants From Plasma in Patients With Non-Small Cell Lung Cancer.

JCO clinical cancer informatics·2025
Same author

Evaluation of the ASPYRE-Lung targeted variant panel: a rapid, low-input solution for non-small cell lung cancer biomarker testing and experience from three independent sites.

Translational lung cancer research·2024
Same author

ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Frontiers in oncology·2024
Same author

The hagfish genome and the evolution of vertebrates.

Nature·2024

Related Experiment Video

Updated: Jun 2, 2026

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
13:24

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

Published on: April 11, 2016

A method for counting PCR template molecules with application to next-generation sequencing.

James A Casbon1, Robert J Osborne, Sydney Brenner

  • 1Population Genetics Technologies Ltd., Babraham Institute, Babraham, Cambridgeshire CB22 3AT, UK.

Nucleic Acids Research
|April 15, 2011
PubMed
Summary
This summary is machine-generated.

This study introduces a simple method using degenerate bases to count DNA template molecules after polymerase chain reaction (PCR) amplification. This technique improves genotyping accuracy and reduces errors in next-generation sequencing library preparation.

More Related Videos

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
10:24

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Published on: August 29, 2014

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing
12:33

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Published on: July 28, 2017

Related Experiment Videos

Last Updated: Jun 2, 2026

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
13:24

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

Published on: April 11, 2016

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
10:24

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Published on: August 29, 2014

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing
12:33

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Published on: July 28, 2017

Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Polymerase chain reaction (PCR) amplification is crucial for preparing DNA templates for next-generation sequencing (NGS).
  • PCR amplification, while increasing DNA molecule numbers, can alter the original template representation and introduce random errors.
  • These alterations impede accurate quantification essential for applications like allele calling and microbial diversity estimation.

Purpose of the Study:

  • To develop a straightforward method for counting DNA template molecules following PCR amplification.
  • To enhance the accuracy of genotyping and reduce PCR-induced noise.
  • To improve the reliability of variant calling in sequencing data.

Main Methods:

  • A novel method utilizing degenerate bases to quantify template DNA molecules was developed.
  • The method was integrated into existing DNA library preparation workflows.
  • The impact on PCR amplification noise and genotyping accuracy was assessed.

Main Results:

  • The degenerate base method effectively counts template molecules, improving genotyping accuracy.
  • The technique successfully mitigates noise introduced by PCR amplification.
  • Implementation of this method enhances the precision of variant calling.

Conclusions:

  • This simple, adaptable method using degenerate bases offers a significant improvement for PCR-based DNA quantification.
  • It addresses key limitations in NGS library preparation, leading to more accurate downstream analyses.
  • The technique holds promise for applications demanding precise DNA quantification, including genetic variant detection and microbiome studies.