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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....

Sanger Sequencing

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-seq

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.
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PCR

PCR

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.
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Related Experiment Video

Updated: Nov 20, 2025

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

Published on: January 12, 2021

PCR Techniques in Next-Generation Sequencing.

Rashmi S Goswami¹

¹Department of Laboratory Hematology, University Health Network, Toronto General Hospital, 200 Elizabeth Street, Room 11EB-453, Toronto, ON, M5G 2C4, Canada. Goswami.rashmi@gmail.com.

Methods in Molecular Biology (Clifton, N.J.)

|February 5, 2016

Summary

This summary is machine-generated.

Polymerase chain reaction (PCR) remains crucial for next-generation sequencing (NGS) in clinical diagnostics. PCR enables efficient targeted sequencing of cancer mutation hotspots, overcoming challenges of whole-genome sequencing.

Keywords:

AmpliSeq Hotspot mutation panel Ion Torrent Multiplex PCR Next-generation sequencing Targeted sequencing

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Last Updated: Nov 20, 2025

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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Published on: August 29, 2014

Area of Science:

Molecular diagnostics
Genomics
Cancer research

Background:

Next-generation sequencing (NGS) is increasingly used in clinical settings.
Whole genome sequencing faces challenges in clinical implementation due to cost and data management.
Targeted sequencing offers increased efficiency for clinical diagnostics.

Purpose of the Study:

To highlight the continued importance of PCR in NGS.
To describe PCR methods for targeted genomic region amplification.
To focus on cancer mutation hotspots for clinical applications.

Main Methods:

Utilizing Polymerase Chain Reaction (PCR) techniques.
Employing targeted sequencing strategies.
Using the Ampliseq Cancer Hotspot v2 panel for amplification.

Main Results:

PCR is integral to the success of targeted NGS.
PCR facilitates the generation of multiple NGS libraries.
Simultaneous sequencing of multiple targeted regions is enabled by PCR.

Conclusions:

PCR remains essential for efficient clinical NGS.
Targeted sequencing via PCR is a viable approach for cancer mutation detection.
The described PCR methods support clinical molecular diagnostics.