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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....

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Related Experiment Video

Updated: Dec 29, 2025

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Published on: October 16, 2018

Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

Jennifer Chiniquy^1,2, Megan E Garber^1,3, Aindrila Mukhopadhyay¹

¹Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.

|January 30, 2020

Summary

This study introduces fluorescent amplification for next generation sequencing (FA-NGS) library preparation, streamlining workflows with real-time quantitative PCR (qPCR) for faster, more accurate results. The FA-NGS method reduces steps and improves quality control, minimizing user error in high-throughput sequencing.

Keywords:

Echo High-throughput Library preparation NGS Next generation sequencing SYBR green qPCR

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Last Updated: Dec 29, 2025

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Published on: October 16, 2018

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

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Published on: August 29, 2014

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

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Published on: March 22, 2016

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Next-generation sequencing (NGS) library preparation is labor-intensive and involves end-of-process quality control (QC).
Increasing throughput exacerbates challenges with conventional multiplexed library preparation.

Purpose of the Study:

To develop a streamlined, high-throughput NGS workflow.
To implement single-tube, single-reagent QC during library preparation.

Main Methods:

Modified conventional workflows by replacing PCR and quantification with real-time quantitative PCR (qPCR) using SYBR Green I.
Implemented melting curve analysis for intermediate QC.
Developed a software toolkit for data analysis and pooling instructions.

Main Results:

qPCR enabled direct, single-tube library quantification and pooling without extra reagents.
Melting curve analysis confirmed successful amplification.
Sequencing data showed even representation of indexed libraries, validating qPCR-based pooling calculations.
FA-NGS was successfully applied to plasmid and bacterial genome libraries.

Conclusions:

The FA-NGS workflow reduces overall steps and risk of user error.
Melting curve analysis effectively identifies library failures before sequencing.
FA-NGS shows versatility for various library types beyond plasmids and bacterial genomes.