100重微单倍型复合检测体系建立及其在BGI-500RS和MiSeq测序平台的应用

Development of a 100-plex microhaplotype panel and its application on BGI-500RS and MiSeq sequencing platforms

目的 建立一个在BGI-500RS和MiSeq测序平台均适用的微单倍型复合检测体系,评估此微单倍型体系在两个测序平台的检测效能。方法 针对100个微单倍型基因座设计扩增引物并优化复合检测体系,分别采用扩增后酶连接头的方法实现BGI-500RS和MiSeq双平台的文库构建,对11名个体DNA样本分别在两个平台进行测序。结果 本研究建立了一个包含100个微单倍型基因座的复合检测体系,该体系在BGI-500RS和MiSeq测序平台均适用。利用本体系在BGI-500RS和MiSeq平台检测11份样本,97%以上的基因座在两平台均可稳定检出。两个平台所有杂合子基因座的平均等位基因覆盖率都在0.7以上,说明基因座内均衡性良好。同一样本在两平台均检出的基因座的分型一致性达100%。结论 本研究建立了一个包含100个微单倍型基因座的复合扩增体系,该体系在BGI-500RS和MiSeq测序平台检测结果的均衡性、稳定性相当。相同样本在两个平台均检出的基因座分型完全一致,测序深度的差异源自测序平台的通量差异。

Objective To develop a multiplex microhaplotype panel on BGI-500RS and Mi Seq sequencing platforms and evaluate the detection efficiency of this panel on these two sequencing platforms. Methods The amplification primers were designed for 100 microhaplotype loci and the multiplex detection panel was optimized. Library preparation on BGI-500RS and Mi Seq platforms were achieved by enzymatic ligation of adapters after amplifications. Then 11 samples were sequenced on the two platforms, respectively. Results In this study, a multiplex amplification panel containing 100 microhaplotype loci was developed, which was applicable to both BGI-500RS and Mi Seq sequencing platforms. Eleven samples were detected with this panel and more than 97 % of the loci can be stably detected on the two sequencing platforms. The average allele coverage ratios of all heterozygous loci on both sequencing platforms were above 0.7, indicating satisfactory intra-locus balances.The genotyping consistency of the loci detected from the same samples on the both platforms was 100 %. Conclusion A microhaplotype panel containing 100 microhaplotype loci was developed in this study. Equivalent intra-locus balance as well as stability was observed for the 100-plex microhaplotype panel on BGI-500RS and MiSeq sequencing platforms. The genotyping results of the same samples detected on the two platforms were completely consistent. The reason for significantly different sequencing depths of the microhaplotype markers was the different throughputs of the sequencing platforms.