Table3_The application of short and highly polymorphic microhaplotype loci in paternity testing and sibling testing of temperature-dependent degraded samples.XLSX

Paternity testing and sibling testing become more complex and difficult when samples degrade. But the commonly used genetic markers (STR and SNP) cannot completely solve this problem due to some disadvantages. The novel genetic marker microhaplotype proposed by Kidd’s research group combines the advantages of STR and SNP and is expected to become a promising genetic marker for kinship testing in degraded samples. Therefore, in this study, we intended to select an appropriate number of highly polymorphic SNP-based microhaplotype loci, detect them by the next-generation sequencing technology, analyze their ability to detect degraded samples, calculate their forensic parameters based on the collected 96 unrelated individuals, and evaluate their effectiveness in paternity testing and sibling testing by simulating kinship relationship pairs, which were also compared to 15 STR loci. Finally, a short and highly polymorphic microhaplotype panel was developed, containing 36 highly polymorphic SNP-based microhaplotype loci with lengths smaller than 100 bp and Ae greater than 3.00, of which 29 microhaplotype loci could not reject the Hardy-Weinberg equilibrium and linkage equilibrium after the Bonferroni correction. The CPD and CPE of these 29 microhaplotype loci were 1-2.96E-26 and 1-5.45E-09, respectively. No allele dropout was observed in degraded samples incubated with 100°C hot water for 40min and 60min. According to the simulated kinship analysis, the effectiveness at the threshold of 4/−4 reached 98.39% for relationship parent-child vs. unrelated individuals, and the effectiveness at the threshold of 2/−2 for relationship full-sibling vs. unrelated individuals was 93.01%, which was greater than that of 15 STR loci (86.75% for relationship parent-child vs. unrelated individuals and 81.73% for relationship full-sibling vs. unrelated individuals). After combining our 29 microhaplotype loci with other 50 short and highly polymorphic microhaplotype loci, the effectiveness values at the threshold of 2/−2 were 82.42% and 90.89% for relationship half-sibling vs. unrelated individuals and full-sibling vs. half-sibling. The short and highly polymorphic microhaplotype panel we developed may be very useful for paternity testing and full sibling testing in degraded samples, and in combination with short and highly polymorphic microhaplotype loci reported by other researchers, may be helpful to analyze more distant kinship relationships.

当样本发生降解时，亲子鉴定与同胞鉴定的难度与复杂度均会显著提升。但当前常用的遗传标记短串联重复序列（Short Tandem Repeat, STR）与单核苷酸多态性（Single Nucleotide Polymorphism, SNP）因自身存在一定缺陷，无法完全解决此类问题。Kidd研究团队提出的新型遗传标记微单倍型（microhaplotype）兼具STR与SNP的优势，有望成为降解样本亲缘关系鉴定的理想遗传标记。因此，本研究拟筛选适量多态性较高的基于SNP的微单倍型位点，采用下一代测序技术（Next-Generation Sequencing, NGS）对其进行检测，分析其对降解样本的适配能力；基于收集的96名无亲缘关系个体的样本，计算该类位点的法医遗传学参数；通过模拟亲缘关系对评估其在亲子鉴定与同胞鉴定中的效能，并与15个STR位点的检测效能进行对比。最终，本研究构建了一套短小且多态性优异的微单倍型检测体系，包含36个长度小于100 bp、有效等位基因数（effective number of alleles, Ae）大于3.00的基于SNP的高多态性微单倍型位点；其中29个位点经Bonferroni校正后，符合哈迪-温伯格平衡（Hardy-Weinberg equilibrium, HWE）与连锁平衡（linkage equilibrium, LE）。该29个微单倍型位点的累计个人识别能力（Combined Power of Discrimination, CPD）与累计非父排除概率（Combined Power of Exclusion, CPE）分别为1 - 2.96×10^-26与1 - 5.45×10^-9。经100℃水浴处理40分钟与60分钟的降解样本中，未检测到等位基因脱扣（Allele Dropout, ADO）现象。模拟亲缘关系分析结果显示，在判定阈值为4/−4时，该体系对“父母子-无关个体”亲缘关系的识别准确率达98.39%；在判定阈值为2/−2时，对“全同胞-无关个体”亲缘关系的识别准确率为93.01%，均优于15个STR位点（15个STR位点对“父母子-无关个体”的识别准确率为86.75%，对“全同胞-无关个体”的识别准确率为81.73%）。将本研究的29个微单倍型位点与其他50个短小高多态性微单倍型位点合并后，在判定阈值为2/−2时，该组合对“半同胞-无关个体”与“全同胞-半同胞”亲缘关系的识别准确率分别为82.42%与90.89%。本研究构建的短小高多态性微单倍型检测体系，可有效应用于降解样本的亲子鉴定与全同胞鉴定；若与其他研究者报道的短小高多态性微单倍型位点联合使用，则可辅助分析更远缘的亲缘关系。