基于多重PCR靶向二代测序的近交系小鼠遗传质量监测方法建立
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摘要
目的建立基于多重PCR靶向二代测序的小鼠遗传质量监测方案。方法从小鼠单核苷酸多态性(SNP)数据库筛选出相对均匀分布在20条染色体上的112个SNP位点,然后对SNP位点附近片段进行多重PCR扩增,建库后进行Illumina高通量测序,对原始测序数据进行生物信息学分析获得SNP信息。结果测序结果显示,多重PCR的扩增子均一性好,各片段成功率高达90%以上,特异性高,高深度测序条件下, SNP位点的等位基因比例趋近于1或0,符合纯合子条件;分析4批近交系小鼠样本,表明SNP位点成功鉴定的比例分别为99.82%, 92.00%, 99.10%和90.35%,且所有小鼠品系个体的SNP位点均为纯合,并被成功确定为目标品系;品系间两两比较,最大差异数为73个,最小差异数为3个,差异位点平均数为53个,差异中位数为60个,显示出本方案对常见近交系小鼠品系分辨率较高。结论多重PCR靶向二代测序方案的SNP分型方案是一种准确、快速、高效的基因分型方案,可用于遗传质量检测和品系鉴定。
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.
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.
引文
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[15]徐伟,晁天柱,刘丽均,等.小鼠冷冻胚胎和精子SNP遗传鉴定方案的建立[J].中国实验动物学报,2016,24(2):169-174.
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[2]Petkov PM,Cassell MA,Sargent EE,et al.Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse[J].Genomics,2004,83(5):902-911.
[3]Chen K,Zhou YX,Li K,et al.Multiplex PCR with the blunt hairpin primers for next generation sequencing[J].Biotechnol Bioproc E,2017,22:347-351.
[4]谢雯,鲍世民,谢建云,等.基于PCR-LDR平台的近交系小鼠遗传质量快速检测方法[J].中国实验动物学报,2012,20(4):1-8.
[5]Chen K,Zhou YX,Li K,et al.A novel three-roundmultiplex PCR for SNP genotyping with next generation sequencing[J].Anal Bioanal Chem,2016,408(16):4371-4377.
[6]Bioinformatics B FastQC:a quality control tool for high throughput sequence data[DB].http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.2014.
[7]Hannon Lab FASTX-Toolkit,FASTQ/A short-reads preprocessing tools[DB].http://hannonlab.cshl.edu/fastx_toolkit/index.html.Accessed 25 Feb 2011.
[8]Marcel M.Cutadapt removes adapter sequences from highthroughput sequencing reads[J].EMBnet.journal,2011,17(1):10-12.
[9]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler Transform[J].Bioinformatics,2009,25(14):1754-1760.
[10]Li H,Handsaker B,Wysoker A,et al.The Sequence alignment/map(SAM)format and SAMtools[J].Bioinformatics,2009,25(16):2078-2079.
[11]李凯,谢雯,鲍世民,等.一种用于鉴别近交系小鼠的SNP分型方法[P].中国发明专利(授权),2014.ZLGL2012090102.
[12]朱福龄,王汉荣,黄帼英,等.应用多价抗血清作为近交系小鼠遗传控制检测方法的探讨[J].实验动物与比较医学,1999,6(2):99-101.
[13]陈丙波,魏泓,罗刚,等.应用α-珠蛋白3'-HVR探针进行小鼠的DNA指纹分析[J].第三军医大学学报,1999,21(2):97-99.
[14]陈振文.DNA指纹图与微卫星DNA技术在近交系大、小鼠遗传监测中的应用研究[D].北京:中国农业大学,2004.
[15]徐伟,晁天柱,刘丽均,等.小鼠冷冻胚胎和精子SNP遗传鉴定方案的建立[J].中国实验动物学报,2016,24(2):169-174.
[16]Xu FY,Chao TZ,Zhang Y,et al.Chromosome 1 sequence analysis of C57BL/6J-Chr1KM mouse strain[J].Int Jgenomics,2017,2017:1-7.
[17]李银银,吴绍亮,王洪,等.微卫星DNA分析国内24个近交系小鼠遗传状况[J].中国比较医学杂志,2017,28(3):43-49.