利用分子特异的条形码评估二代测序中STR的复制滑脱

Eevaluation of STR stutters caused by strand slippage during replication in second generation sequencing with unique molecular identifiers

目的利用分子特异的条形码(Unique Molecular Identifiers,UMIs)研究二代测序平台中STR的复制滑脱现象。方法采用探针捕获与高通量测序技术对12个中国汉族无关个体进行STR靶标基因座的富集与测序,并利用自己开发的软件进行STR分型及复制滑脱的评估。结果对12个中国汉族无关个体的高通量测序数据的STR分型及复制滑脱情况的分析发现:单个UMI家族随着成员数目的增多,其STR的复制滑脱产物占所有成员的百分比(Stutter Percentage,SP)逐渐降低,当家族成员数目增至40个时,其SP趋于稳定;STR基因座的各等位基因易向比它自身少一个重复单位的等位基因滑脱,形成stutters。结论UMI可准确识别STR基因座在PCR扩增过程中DNA链滑脱所产生的stutters并将其去除,达到了降低二代测序平台中STR复制滑脱产物的效果,为法医鉴定中混合检材的精细鉴别提供了技术支撑。

Objective To study the stutters caused by strand slippage of STR during PCR amplification in the second generation sequencing platform using unique molecular identifiers(UMIs).Methods Probe capture and highthroughput sequencing technology were used to enrich and sequence targeted STR loci in 12 unrelated Chinese Han individuals,and the self-developed software was used for STR genotyping and the evaluation of STR slippage phenomenon in the second generation sequencing platform.Results STR genotyping and slippage phenomenon on the high-throughput sequencing data of 12 unrelated Chinese Han individuals showed that with the increase of the number of members for a single UMI family,the percentage of stutters(SP)produced during STR replication in all members of the family gradually decreased;when the number of members increased to 40,its SP began to be stable.The major alleles of most STR loci tended to slip to the alleles with one less repeat unit than itself.Conclusion UMIs can accurately identify and remove stutters produced by STR loci in the PCR amplification,achieve the effect of reducing strand slippage during STR replication on the second generation sequencing platform,and provide technical support for the fine identification of mixed samples in forensic medicine.