A novel three-round multiplex PCR for SNP genotyping with next generation sequencing.
Owing to the high throughput and low cost, next generation sequencing has attracted much attention for SNP genotyping application for researchers. Here, we introduce a new method based on three-round multiplex PCR to precisely genotype SNPs with next generation sequencing. This method can as much as possible consume the equivalent amount of each pair of specific primers to largely eliminate the amplification discrepancy between different loci. After the PCR amplification, the products can be directly subjected to next generation sequencing platform. We simultaneously amplified 37 SNP loci of 757 samples and sequenced all amplicons on ion torrent PGM platform; 90.5 % of the target SNP loci were accurately genotyped (at least 15×) and 90.4 % amplicons had uniform coverage with a variation less than 50-fold. Ligase detection reaction (LDR) was performed to genotype the 19 SNP loci (as part of the 37 SNP loci) with 91 samples randomly selected from the 757 samples, and 99.5 % genotyping data were consistent with the next generation sequencing results. Our results demonstrate that three-round PCR coupled with next generation sequencing is an efficient and economical genotyping approach. Graphical Abstract The schematic diagram of three-round PCR.
College of Environmental Science and Engineering, Donghua University, Shanghai, 05003365, China.
Ke
K
Chen
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 05003365, China.
Yu-Xun
YX
Zhou
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 05003365, China.
Kai
K
Li
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 05003365, China.
Li-Xin
LX
Qi
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 05003365, China.
Qi-Fei
QF
Zhang
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 05003365, China.
Mao-Chun
MC
Wang
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 05003365, China. xiaojunhua@dhu.edu.cn.
Jun-Hua
JH
Xiao
D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015894:Genome, Human; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D004247:DNA
Next generation sequencing; SNP genotyping; Three-round multiplex PCR