Data from: HiMAP: robust phylogenomics from highly multiplexed amplicon sequencing

High-throughput sequencing has fundamentally changed how molecular phylogenetic datasets are assembled, and phylogenomic datasets commonly contain 50-100-fold more loci than those generated using traditional Sanger-based approaches. Here, we demonstrate a new approach for building phylogenomic datasets using single tube, highly multiplexed amplicon sequencing, which we name HiMAP (Highly Multiplexed Amplicon-based Phylogenomics), and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and post-sequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously, requires minimal hands-on time at the bench (<1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically-important agricultural insect pests. The resulting filtered dataset (>150,000 bp concatenated alignment, ~20% missing character sites across all individuals and amplicons) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting dataset provides into relationships of this diverse insect group.

高通量测序从根本上重塑了分子系统发育数据集的构建范式，系统发育基因组数据集所包含的基因座数量通常是传统桑格测序法所得数据集的50至100倍。本研究提出一种基于单管高通量多重扩增子测序的系统发育基因组数据集构建新方法，将其命名为HiMAP（Highly Multiplexed Amplicon-based Phylogenomics，基于多重扩增子的系统发育基因组学），同时提供了基于基因组与转录组数据资源进行基因座筛选，以及测序后共识序列调用与序列比对的生物信息学分析流程。该方法成本低廉，可同时对数百个分类单元开展测序，实验室操作耗时极短（不足半天），且数据分析无需进行读段比对或序列组装。本研究通过单管反应对覆盖7个属的82种实蝇科果蝇（共384个个体）的878个扩增子进行测序，验证了该方法的有效性，其中包含多种经济危害严重的农业害虫。经筛选后的最终数据集（拼接比对序列长度超过150,000 bp，所有个体与扩增子的特征位点缺失率约为20%）包含超过40,000个系统发育信息位点；尽管不同分析间存在一定的拓扑冲突，但该数据集为该类群的诸多系统发育关系提供了前所未有的分辨率。尤为重要的是，本研究发现强有力的支持证据证实了凹唇实蝇属（Zeugodacus）的分类地位，以及果实蝇属（Dacus）与凹唇实蝇属（Zeugodacus）的姐妹群关系。本研究从分子与生物信息学优势的角度对HiMAP进行了探讨，并阐述了该数据集为这一多样昆虫类群的系统发育关系研究提供的全新认知。