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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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Updated: Feb 25, 2026

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Published on: January 30, 2019

Next-generation sequencing-based analysis of reverse transcriptase fidelity.

Kiyoshi Yasukawa¹, Kei Iida², Hiroyuki Okano¹

¹Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

Biochemical and Biophysical Research Communications

|August 6, 2017

Summary

This summary is machine-generated.

We developed a fast, simple next-generation sequencing method to measure reverse transcriptase (RT) fidelity. This technique accurately quanties RT error rates, crucial for molecular biology applications.

Keywords:

Error rate Fidelity NGS Reverse transcriptase cDNA

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Last Updated: Feb 25, 2026

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

Published on: June 16, 2011

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Accurate reverse transcriptase (RT) activity is essential for molecular biology techniques like cDNA synthesis.
Existing methods for assessing RT fidelity can be complex and time-consuming.
High-throughput, precise evaluation of RT error rates is needed.

Purpose of the Study:

To develop a simple, rapid, and high-throughput method for analyzing reverse transcriptase fidelity using next-generation sequencing (NGS).
To accurately quantify mutation rates introduced by different reverse transcriptases during cDNA synthesis.

Main Methods:

A novel method combining cDNA synthesis with a tagged primer, PCR amplification using high-fidelity DNA polymerase, and NGS.
Sequence analysis to identify mutations by comparing reads to a reference sequence.
Distinguishing errors originating from cDNA synthesis, PCR, or NGS based on tagged sequence reads.

Main Results:

The method successfully identified and quantified mutations introduced by reverse transcriptases.
Error rates for Moloney murine leukemia virus (MMLV) RT variant MM4 and Thermococcus kodakarensis RTX were 0.75-1.0 × 10⁻⁴ errors/base.
Wild-type human immunodeficiency virus type 1 (HIV-1) RT showed a higher error rate of 2.6 × 10⁻⁴ errors/base.

Conclusions:

The developed NGS-based method provides a precise and high-throughput evaluation of reverse transcriptase fidelity.
This technique does not require expensive equipment or complex procedures like adaptor ligation.
The method is versatile for assessing various RTs under different reaction conditions.