Schmidt Alexandra, Schneider Clément, Decker Peter, Hohberg Karin, Römbke Jörg, Lehmitz Ricarda, Bálint Miklós
Senckenberg Biodiversity Climate Research Center Frankfurt am Main Germany.
Biology Department J.W. Goethe University Frankfurt am Main Germany.
Ecol Evol. 2022 Jun 6;12(6):e8991. doi: 10.1002/ece3.8991. eCollection 2022 Jul.
Metagenomics - shotgun sequencing of all DNA fragments from a community DNA extract - is routinely used to describe the composition, structure, and function of microorganism communities. Advances in DNA sequencing and the availability of genome databases increasingly allow the use of shotgun metagenomics on eukaryotic communities. Metagenomics offers major advances in the recovery of biomass relationships in a sample, in comparison to taxonomic marker gene-based approaches (metabarcoding). However, little is known about the factors which influence metagenomics data from eukaryotic communities, such as differences among organism groups, the properties of reference genomes, and genome assemblies.We evaluated how shotgun metagenomics records composition and biomass in artificial soil invertebrate communities at different sequencing efforts. We generated mock communities of controlled biomass ratios from 28 species from all major soil mesofauna groups: mites, springtails, nematodes, tardigrades, and potworms. We shotgun sequenced these communities and taxonomically assigned them with a database of over 270 soil invertebrate genomes.We recovered over 95% of the species, and observed relatively high false-positive detection rates. We found strong differences in reads assigned to different taxa, with some groups (e.g., springtails) consistently attracting more hits than others (e.g., enchytraeids). Original biomass could be predicted from read counts after considering these taxon-specific differences. Species with larger genomes, and with more complete assemblies, consistently attracted more reads than species with smaller genomes. The GC content of the genome assemblies had no effect on the biomass-read relationships. Results were similar among different sequencing efforts.The results show considerable differences in taxon recovery and taxon specificity of biomass recovery from metagenomic sequence data. The properties of reference genomes and genome assemblies also influence biomass recovery, and they should be considered in metagenomic studies of eukaryotes. We show that low- and high-sequencing efforts yield similar results, suggesting high cost-efficiency of metagenomics for eukaryotic communities. We provide a brief roadmap for investigating factors which influence metagenomics-based eukaryotic community reconstructions. Understanding these factors is timely as accessibility of DNA sequencing and momentum for reference genomes projects show a future where the taxonomic assignment of DNA from any community sample becomes a reality.
宏基因组学——对群落DNA提取物中的所有DNA片段进行鸟枪法测序——通常用于描述微生物群落的组成、结构和功能。DNA测序技术的进步以及基因组数据库的可用性，越来越使得在真核生物群落中使用鸟枪法宏基因组学成为可能。与基于分类标记基因的方法（元条形码技术）相比，宏基因组学在恢复样本中的生物量关系方面取得了重大进展。然而，对于影响真核生物群落宏基因组学数据的因素，如生物类群之间的差异、参考基因组的特性以及基因组组装，我们了解得还很少。我们评估了在不同测序深度下，鸟枪法宏基因组学如何记录人工土壤无脊椎动物群落的组成和生物量。我们从所有主要土壤中型动物类群的28个物种中生成了具有可控生物量比例的模拟群落，这些类群包括螨类、弹尾虫、线虫、缓步动物和线蚓。我们对这些群落进行了鸟枪法测序，并使用一个包含超过270个土壤无脊椎动物基因组的数据库对它们进行分类。我们鉴定出了超过95%的物种，并观察到相对较高的假阳性检测率。我们发现分配给不同分类群的 reads 存在很大差异，一些类群（如弹尾虫）始终比其他类群（如线蚓）吸引更多的 hits。在考虑了这些分类群特异性差异后，可以根据 reads 计数预测原始生物量。基因组较大且组装更完整的物种始终比基因组较小的物种吸引更多的 reads。基因组组装的GC含量对生物量与reads的关系没有影响。不同测序深度的结果相似。结果表明，从宏基因组序列数据中恢复分类群和生物量恢复的分类群特异性存在相当大的差异。参考基因组和基因组组装的特性也会影响生物量恢复，在真核生物的宏基因组研究中应予以考虑。我们表明，低测序深度和高测序深度产生的结果相似，这表明宏基因组学对真核生物群落具有很高的成本效益。我们提供了一个简要的路线图，用于研究影响基于宏基因组学的真核生物群落重建的因素。随着DNA测序的可及性和参考基因组项目的推进，表明未来从任何群落样本中进行DNA分类鉴定都将成为现实，了解这些因素正当时。
