Genetic Structure of Duplicated Sequences Revealed by Genotyping Single Sperm. Homo sapiens

Three different experimental approaches were evaluated for discrimination of genomic variance in and between duplicated sequences using 48 markers in duplicon regions and 17 SNPs in unique sequences previously characterized in another study. We found only the method high-throughput single sperm typing could conclusively resolve the alleles of all markers. Resulting data from single sperm analysis were also used to examine the genetic structure of duplicon markers in the human population. Single sperm typing can be a rapid, efficient and accurate method for initial screening and assessment of genetic variation and for detailed genetic analysis of duplicon markers. Keywords: Genotyping Overall design: Sixty-five markers including 17 MSVs, 12 PSVs, 19 SIDs and 17 SNPs in unique sequences described in Fredman et al. were selected for study. The samples include 40 genomic DNA samples from four ethnic groups, semen samples from 11 donors, and 10 to 20 sperm from each donor except one, AB012, for whom 65 sperm were analyzed. Both genomic and sperm DNA samples were subject to multiplex amplification followed by microarray analysis. Genotypes were determined by using the Accutyping software. Semen samples were genotyped on both strands. Allele status in these samples were compared and analyzed. The single sperm typing method allowed us to identify markers residing in non-unique sequence, to analyze the detailed genetic structure of the duplicons and to learn whether different alleles are present for the duplicon sequences in the human population.

本研究利用此前另一项研究中已完成表征的48个重复拷贝序列（duplicon）区域标记与17个独特序列单核苷酸多态性（Single Nucleotide Polymorphism, SNP），对三种不同实验方法在区分重复序列内部及重复序列间基因组变异的效能展开评估。结果显示，唯有高通量单精子分型（high-throughput single sperm typing）技术能够明确分辨所有标记的等位基因。单精子分析所得数据还被用于探究人类群体中重复拷贝序列标记的遗传结构。单精子分型技术可作为一种快速、高效且精准的方法，用于遗传变异的初筛与评估，以及重复拷贝序列标记的精细遗传分析。

关键词：基因分型（Genotyping）
整体实验设计：本研究选取了65个标记，其中包括Fredman等人报道的独特序列区域内的17个MSVs、12个PSVs、19个SIDs以及17个单核苷酸多态性（SNP）。实验样本涵盖4个族群的40份基因组DNA样本、11名捐献者的精液样本；除捐献者AB012外，其余每名捐献者的精子样本均采集10~20个精子进行分析，AB012则共分析了65个精子。基因组DNA与精子DNA样本均先进行多重扩增，随后开展微阵列分析。基因型通过Accutyping软件进行判定。精液样本的基因分型检测覆盖DNA双链。对上述样本中的等位基因状态进行了比较与分析。单精子分型技术使得本研究得以鉴定位于非独特序列区域的标记，解析重复拷贝序列的精细遗传结构，并探明人类群体中重复拷贝序列是否存在不同的等位基因。