毛细管电泳联合二代测序技术用于亲子鉴定突变分析

Next Generation Sequencing Coalesced into Capillary Electrophoresis to Analyze Mutations from Paternity Testing

目的联合应用毛细管电泳与二代测序技术,探索肯定亲权案件中的STR基因座的突变率和突变方式。方法2600例肯定亲权关系的案件材料采用PowerPlex21试剂盒进行STR检验,发现67例存在基因座突变,对该67例亲子关系(62例三联体,5例二联体)的196个样本用SeqTyper®24构建文库,使用Ion PGMTM平台进行二代测序。结果12个STR基因座共发现71个突变,父源突变与母源突变的比例为3.13‥1。其中64例亲子关系观察到1个基因座突变,2例观察到2个基因座突变,1例观察到3个基因座突变,有些突变从毛细管电泳所得STR基因座结果无法判断是增加还是减少了一步,二代测序则可以明晰等位基因的遗传方式及突变情况。结论对于复杂核心序列、含不完全重复单位的基因座,有些突变可以通过二代测序明确突变的来源和方式,更直观地从微观碱基序列的角度观察等位基因的遗传。

Objective To coalesce capillary electrophoresis into next generation sequencing for exploring the mutational patterns among STR loci from paternity testing.Methods From 2600 cases of confirmed paternity,the relevant STR loci were screened with PowerPlex21 kit,thus having 67 cases found of mutations that involved with 196 samples(relating to 62 trios and 5 duos)which were afterwards detected through SeqTyper®24 to construct the correlative library.Further,Ion PGMTM platform was adopted to carry out next generation sequencing.Results There were 12 STR loci having been found of totaling 71 mutations,among which the mutant ratio of paternal to maternal was 3.13:1.Single one-locus mutations were observed in 64 cases,with the two-locus’in two cases and the three-locus’in one case.For some mutations,it is difficult to determine whether there has been increased or decreased of a step from the capillary electrophoretic results of STR loci.In contrast,next generation sequencing can clarify the inheritance route and mutational pattern.Conclusion For the locus harboring complex core sequence and/or incomplete repetitive unit,next generation sequencing is able to identify and confirm certain mutations,having the inheritance of alleles observed more directly from the microscopic DNA base sequences.