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A multiplex primer design algorithm for target amplification of continuous genomic regions.

Ahmet Rasit Ozturk¹, Tolga Can²

¹Middle East Technical University, Informatics Institute, Ankara, Turkey. ahmetrasit@gmail.com.

BMC Bioinformatics

|June 21, 2017

Summary

We developed a novel Multiplex PCR (MPCR) primer design method for efficient Next Generation Sequencing (NGS) assays. This approach streamlines amplicon preparation, offering a reliable solution for targeted sequencing of smaller genomic regions.

Keywords:

Multiplex PCR Next Generation sequencing Primer design Target amplification

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Targeted Next Generation Sequencing (NGS) offers cost-efficiency and reliability over Sanger sequencing.
Target enrichment is suitable for large gene sets, while target amplification is more efficient for smaller genomic regions.
Multiplex PCR (MPCR) addresses challenges in amplicon preparation for target amplification, reducing time, equipment, and labor.

Purpose of the Study:

To propose a novel method for designing Multiplex PCR (MPCR) primers for continuous genomic regions.
To optimize primer design for reliable and efficient Next Generation Sequencing (NGS) assays.
To address the difficulties associated with amplicon preparation in target amplification methods.

Main Methods:

Development of a novel MPCR primer design algorithm.
Adherence to clinically reliable PCR design best practices.
Experimental validation using a setup with 48 factor combinations to assess primer design parameters.

Main Results:

The proposed method successfully generated MPCR primer designs for specific genomic regions (HBB gene, MEFV coding regions, human exons).
Benchmarking demonstrated that increasing the initial primer candidate selection sequence length improves results.
Waiting longer for the first feasible solution did not significantly impact outcomes.

Conclusions:

The developed MPCR primer design approach is effective for generating reliable NGS assay primers.
The method provides a solution for targeted sequencing of smaller genomic regions efficiently.
The MPCR strategy enables the production of reliable NGS primers within a reasonable timeframe.