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A novel three-round multiplex PCR for SNP genotyping with next generation sequencing
Ke Chen1, Yu-Xun Zhou2, Kai Li2
1College of Environmental Science and Engineering, Donghua University, Shanghai, 05003365, China.
Analytical and Bioanalytical Chemistry
|April 27, 2016
Summary
This study presents a novel three-round multiplex PCR method for precise single nucleotide polymorphism (SNP) genotyping using next-generation sequencing (NGS). This efficient and economical approach ensures accurate SNP data for large-scale genetic studies.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Next-generation sequencing (NGS) is a powerful tool for SNP genotyping due to its high throughput and low cost.
- Existing methods can suffer from amplification discrepancies between different loci, affecting genotyping accuracy.
Purpose of the Study:
- To introduce and validate a novel three-round multiplex PCR method for precise SNP genotyping.
- To improve amplification uniformity and reduce costs in SNP genotyping using NGS.
Main Methods:
- Developed a three-round multiplex PCR strategy to equalize primer consumption and minimize amplification bias.
- Applied the method to simultaneously amplify 37 SNP loci in 757 samples.
- Sequenced amplicons using the Ion Torrent PGM platform and validated results with Ligase Detection Reaction (LDR).
Main Results:
- Achieved 90.5% accurate genotyping for target SNP loci (≥15× coverage).
- Demonstrated 90.4% uniform amplicon coverage with <50-fold variation.
- Showcased 99.5% concordance between NGS and LDR genotyping for 19 SNP loci in 91 samples.
Conclusions:
- The three-round PCR coupled with NGS is an efficient, economical, and accurate genotyping approach.
- This method effectively addresses amplification discrepancies, enhancing SNP genotyping reliability.
- The technique is suitable for high-throughput and cost-effective genetic analysis.
