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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...

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Related Experiment Video

Updated: May 10, 2026

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Published on: March 22, 2018

Next-generation HLA sequencing using the 454 GS FLX system.

Elizabeth A Trachtenberg¹, Cherie L Holcomb

¹Children's Hospital & Research Center, Oakland, CA, USA.

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

|June 19, 2013

Summary

Next-generation sequencing (NGS) effectively reduces human leukocyte antigen (HLA) genotyping ambiguity. This pyrosequencing method enhances accuracy for complex HLA class I and II loci analysis.

Area of Science:

Immunogenetics
Molecular Biology
Genomics

Background:

Human Leukocyte Antigen (HLA) loci are highly polymorphic and crucial for immune response.
Accurate HLA genotyping is essential for transplantation and disease association studies.
Traditional methods often face challenges with ambiguity in highly complex HLA regions.

Purpose of the Study:

To detail a Next-Generation Sequencing (NGS) protocol for comprehensive HLA class I and II genotyping.
To evaluate the performance, read depth, and ambiguity reduction capabilities of the 454 Life Sciences GS FLX System for HLA analysis.
To demonstrate the utility of clonal amplicon-based pyrosequencing for resolving HLA genotyping ambiguities.

Main Methods:

Utilized 454 Life Sciences GS FLX System with Titanium chemistry for pyrosequencing.

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

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Last Updated: May 10, 2026

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

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Published on: March 22, 2018

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Employed 14 HLA primer pairs with multiplex identifiers (MIDs) for amplicon-based sequencing.

Processed up to 44 samples per plate using clonal, amplicon-based pyrosequencing.

Applied Conexio Assign ATF 454 software for genotype assignment and ambiguity reduction analysis.

Main Results:

Achieved significant reduction in genotyping ambiguity for complex HLA loci.
Demonstrated efficient sequence performance and adequate read depth using the GS FLX System.
Successfully genotyped HLA class I and II loci (HLA-A, -B, -C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1).

Conclusions:

Clonal Next-Generation Sequencing (NGS) provides a robust method for accurate HLA genotyping.
The described protocol and GS FLX System effectively address challenges in analyzing highly polymorphic HLA regions.
This approach significantly improves reliability in HLA typing for clinical and research applications.