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Next-generation Sequencing

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-PCR

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...

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Related Experiment Video

Updated: Jul 3, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Published on: January 8, 2008

High throughput automated allele frequency estimation by pyrosequencing.

Julie Doostzadeh¹, Shadi Shokralla, Farnaz Absalan

¹The Parkinson's Institute, Sunnyvale, California, United States of America.

|July 17, 2008

Summary

This summary is machine-generated.

We developed a cost-effective pyrosequencing assay for accurate allele frequency determination in large populations. This robust method enhances DNA pool genotyping for genetic association studies.

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Last Updated: Jul 3, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Published on: January 8, 2008

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Pyrosequencing for Microbial Identification and Characterization

Published on: August 22, 2013

Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Pyrosequencing is a real-time DNA sequencing technique based on sequencing-by-synthesis.
This method offers cost-effective and user-friendly applications for genetic analysis.
Accurate allele frequency determination is crucial for large-scale genetic association studies.

Purpose of the Study:

To develop an accurate, robust, and cost-efficient method for determining allele frequencies in large populations.
To optimize pyrosequencing for large-scale genotyping of DNA pools.
To minimize systemic sampling errors and improve accuracy in allele frequency analysis.

Main Methods:

Combined multiple methods to enhance pyrosequencing accuracy and efficiency.
Utilized a general biotin amplification approach.
Replaced dTTP with dATP-α-thio to prevent non-uniform peak amplification.

Main Results:

Demonstrated a robust, cost-effective, accurate, and reproducible assay for large-scale DNA pool genotyping.
The developed assay minimizes systemic sampling errors.
Achieved increased accuracy through modified nucleotide incorporation.

Conclusions:

The newly developed pyrosequencing assay is suitable for large-scale genotyping and allele frequency analysis.
The method offers significant advantages in terms of cost, accuracy, and reproducibility.
Potential software improvements can further enhance allele frequency analysis accuracy.