Department of Genetics, Yale University School of Medicine, New Haven, CT, 06520, USA.
Department of Genetics, Yale University School of Medicine, New Haven, CT, 06520, USA.
Forensic Sci Int Genet. 2020 Jul;47:102275. doi: 10.1016/j.fsigen.2020.102275. Epub 2020 Mar 18.
Microhaplotypes (MH) are comprised of multiple single nucleotide polymorphisms (SNPs) that are located within 300 bases of genomic sequence. Improved tools are needed to facilitate broader application of microhaplotypes in a diverse range of populations and forensic settings. We designed an assay for multiplex sequencing of 90 microhaplotypes (mMHseq) that include 46 MH loci with high Effective Number of Alleles (A) from previous studies [1], and 44 high A MH loci containing between four to fourteen SNPs that were identified from the 1000 Genomes (1KG) Project. The unique design of mMHseq integrates a novel method for multiplex amplification from small DNA amounts, and multiplex sequencing of 48 samples in a single MiSeq run to detect all relevant MH variation. Assay performance was evaluated in a cohort of 156 individuals from seven different world populations from Africa, Asia, and Europe. Three of those populations from East Africa (Chagga, Sandawe, and Zaramo) and one from Eastern Europe (Adygei) had sufficient individuals sequenced by the assay to be included in statistical analyses with the 26 1KG populations. For those 30 populations the mean global average A was 5.08 (range: 2.7-11.54) and mean informativeness for biogeographic variation (I) was 0.30 (range: 0.08-0.70). Eighty-five novel SNPs were detected in 58 of the 90 microhaplotypes. Open-source, web-based software was developed to visualize haplotype phase data for each microhaplotype and individual. Our approach for multiplex microhaplotype sequencing can be customized and expanded as novel loci are being discovered.
微单倍型（MH）由位于基因组序列 300 个碱基内的多个单核苷酸多态性（SNP）组成。需要改进工具，以便在不同人群和法医环境中更广泛地应用微单倍型。我们设计了一种用于 90 个微单倍型（mMHseq）多重测序的检测方法，其中包括来自先前研究的 46 个具有高有效等位基因数（A）的 MH 位点[1]，以及 44 个高 A MH 位点，这些位点含有 4 到 14 个 SNP，是从 1000 基因组计划（1KG）中确定的。mMHseq 的独特设计集成了一种从少量 DNA 中进行多重扩增的新方法，以及在单个 MiSeq 运行中对 48 个样本进行多重测序，以检测所有相关的 MH 变异。在来自非洲、亚洲和欧洲的七个不同世界人群的 156 个人的队列中评估了该检测方法的性能。其中三个来自东非（查加、桑达韦和扎拉莫）的人群和一个来自东欧（阿迪格）的人群的个体测序数量足以通过该检测进行统计分析，并与 26 个 1KG 人群进行比较。对于这 30 个群体，全球平均 A 为 5.08（范围：2.7-11.54），生物地理变异的信息量（I）平均值为 0.30（范围：0.08-0.70）。在 90 个微单倍型中，有 58 个检测到 85 个新的 SNP。开发了开源的基于网络的软件，用于可视化每个微单倍型和个体的单倍型相位数据。我们的多重微单倍型测序方法可以根据新发现的位点进行定制和扩展。
