基于多重长PCR靶向捕获测序技术的高同源SNP鉴定  

Identification of highly homologous SNP based on multiplex long PCR targeted capture sequencing technology

作　　者：王决恒 周宇荀[1] 李凯[1] 肖君华[1] WANG Jueheng;ZHOU Yuxun;LI Kai;XIAO Junhua(School of Chemistry and Bioengineering College of Chemistry,Chemical Engineering and Bioitechnology,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学化学化工与生物工程学院,上海

出　　处：《东华大学学报（自然科学版）》2024年第1期163-170,共8页Journal of Donghua University(Natural Science)

基　　金：国家自然科学基金(31772550);上海市科委基金(17140903102)

摘　　要：为建立一种高同源区段的单核苷酸多态性(SNP)基因分型技术,通过构建本地Blast对SNP所在的200和400 bp区段进行同源性评估,并筛选出高同源区段的SNP。利用第一轮多重长PCR(polymerase chain reaction)捕获329个样本的9个高同源区段SNP所在的长片段,使用纯化后的第一轮PCR产物作为模板进行扩增子建库测序,检测样本共得2 928个SNP位点信息,测序成功率高达98.885 6%。利用Hardy-Weinberg(HWE)法则计算试验研究的9个高同源区段SNP位点的基因频率(p值均大于0.05,符合HWE法则),并与NCBI(national center for biotechnology information)中千人基因组数据库中获取的基因频率相比对,发现二者单碱基基因频率一致(误差限<0.15)。研究表明,利用多重长PCR靶向捕获技术结合二代测序技术为高同源区段的SNP分型提供一个准确、快速、大样本检测方案。To establish a genotyping technique for single nucleotide polymorphism(SNP)in highly homologous segments,the homology of 200 and 400 bp segments of SNP was evaluated by running constructed local Blast,and the highly homologous segments SNP were screened out after evaluation.The first round of multiple long-range polymerase chain reaction(PCR)was used to capture the long fragments of 9 highly homologous segments of SNP in 329 samples.The purified products of the first round of PCR were used as templates for amplicon library construction and sequencing.The results show that 2928 SNP loci are detected in 329 samples,and the success rate of sequencing is as high as 98.8856%.According to the Hardy-Weinberg(HWE)rule principle,the gene frequencies of SNP loci in 9 high homologous segments were calculated(p values were greater than 0.05 conform to HWE rule).Compared with the gene frequencies obtained from thousands of genomes database in national center for biotechnology information(NCBI),it was found that the single base gene frequencies of the two segments were consistent(deviance<0.15).It is shown that the use of multi-long PCR targeted capture technology combined with second-generation sequencing,provided an accurate,rapid and large sample detection solution for SNP typing in highly homologous segments.

关 键 词：SNP分型 高同源区段 多重长PCR靶向捕获技术 高通量测序 

分 类 号：Q3[生物学—遗传学]