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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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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Published on: June 21, 2018

MPD: multiplex primer design for next-generation targeted sequencing.

Thomas S Wingo^1,2,3, Alex Kotlar⁴, David J Cutler⁴

¹Division of Neurology, Atlanta VA Medical Center, Decatur, 30033, GA, USA. thomas.wingo@emory.edu.

BMC Bioinformatics

|January 7, 2017

Summary

This summary is machine-generated.

A new software, Multiplex Primer Design (MPD), automates multiplex PCR primer design for next-generation sequencing. MPD ensures primer uniqueness and compatibility, enabling efficient targeted resequencing of genetic regions associated with traits or diseases.

Keywords:

DNA-sequencing Next-generation sequencing Primer design Targeted resequencing

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Targeted resequencing is a cost-effective method for investigating specific genomic regions linked to traits or diseases.
Multiplex PCR amplification is a key technique for targeted resequencing, but designing compatible primer pools is challenging.
Existing open-source software lacks the ability to design genome-wide unique and compatible multiplex PCR primers for next-generation sequencing.

Purpose of the Study:

To develop an open-source software package for automating the design of multiplex PCR primers for next-generation sequencing.
To ensure primers are unique at a genome-level and efficiently pool compatible primers for targeted resequencing experiments.
To provide a user-friendly platform for designing multiplex PCR experiments for various genomic targets.

Main Methods:

Developed Multiplex Primer Design (MPD) software with a C-implemented core for speed and a hashed genome approach for primer uniqueness.
Integrated MPD with a JavaScript web application for user accessibility (http://multiplexprimer.io).
Validated MPD using genes from genome-wide association studies (GWAS), achieving 90% exonic region coverage.

Main Results:

MPD successfully designed multiplex PCR primer pools, achieving 90% exonic region coverage for GWAS-identified genes under stringent criteria.
Wet-lab validation of 47 primer pools demonstrated high-quality sequencing of ~25Kb, with 99.7% completeness and mean coverage of 300X across 313 samples.
Identified 224 variants, consistent with the expected results from high-quality sequencing data.

Conclusions:

MPD effectively designs multiplex PCR experiments for next-generation sequencing.
The software simplifies the adaptation of targeted resequencing pipelines to new genetic targets as evidence emerges.
MPD offers a robust solution for efficient and accurate multiplex PCR primer design in genomic research.