Laboratory Animal and Comparative Medicine ›› 2019, Vol. 39 ›› Issue (2): 111-117.DOI: 10.3969/j.issn.1674-5817.2019.02.008

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Mouse Genetic Quality Monitoring Method Establishment Based on Next-generation Sequencing through Multiplex PCR

QIAN Qiang1, XU Yuan1, WANG Ya-heng1, ZHOU Yu-xun1, XIAO Jun-hua1, HAN Ling2, BAO Shi-ming3, LI Kai1   

  1. 1. Institute of Biological Science and Biotechnology, Donghua University, Shanghai 201620, China;
    2. Shanghai Tenth People's Hospital, Shanghai 200072, China;
    3. Shanghai Institutes for Biological Sciences, China Academy of Science, Shanghai 2000031, China
  • Received:2018-10-31 Online:2019-04-25 Published:2021-01-29

Abstract: Objective To establish a multiplex PCR targeting next-generation sequencing for mouse genetic quality monitoring. Methods Firstly, 112 single nucleotide polymorphisms (SNPs) on 20 chromosomes were screened from common inbred mice. Then, multiplex PCR amplification was performed on the fragments near the SNPs. After the library was constructed, high-throughput sequencing on Illumina platform was performed. The data was analyzed in a bioinformatics pipeline to obtain SNP information. Results The sequencing results showed that the uniform depth of amplicon was obtained, and the success calling rate of each site was over 90%. Secondly, under high specificity and high-depth sequencing conditions, the allele ratio of SNP sites approached 1 or zero, which is consistent with homozygous conditions. After 4 batches of mouse samples (98 in total) were analyzed, the proportions of SNP sites successfully identified were 99.82%, 92.00%, 99.10% and 90.35%, respectively. All mouse individuals were found to be homozygous and were successfully identified as the target strain. Compared differences between each pair strains, the maximum number of difference was 73, the minimum number was 3, and the average number was 53. The median number of difference was 60, showing our approach has a higher resolution for common inbred mouse strains. Conclusion The SNP typing scheme of the multiple PCR protocol is an accurate, rapid and efficient genotyping program for genetic quality testing and strains identification.

Key words: Multiplex PCR, Single nucleotide polymorphisms (SNPs), Inbred mice

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