Thormar Eiríkur A, Hansen Søren B, Jørgensen Louise von Gersdorff, Limborg Morten T
Globe Institute, Faculty of Health and Medical Sciences, Center for Evolutionary Hologenomics University of Copenhagen Copenhagen K Denmark.
Section for Parasitology and Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences University of Copenhagen Frederiksberg C Denmark.
Ecol Evol. 2024 Sep 17;14(9):e70302. doi: 10.1002/ece3.70302. eCollection 2024 Sep.
Despite a surge in microbiota-focused studies in teleosts, few have reported functional data on whole metagenomes as it has proven difficult to extract high biomass microbial DNA from fish intestinal samples. The zebrafish is a promising model organism in functional microbiota research, yet studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics remain scarce. Thus, a consensus on an appropriate sampling method accurately representing the zebrafish gut microbiota, or any fish species is lacking. Addressing this, we systematically tested four methods of sampling the zebrafish gut microbiota: collection of faeces from the tank, the whole gut, intestinal content, and the application of ventral pressure to facilitate extrusion of gut material. Additionally, we included water samples as an environmental control to address the potential influence of the environmental microbiota on each sample type. To compare these sampling methods, we employed a combination of genome-resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage, and population level microdiversity. Comparison to the environmental control highlighted the potential impact of the environmental contamination on data interpretation. While all sample types tested are informative about the zebrafish gut microbiota, the results show that optimal sample type for studying fish microbiomes depends on the specific objectives of the study, and here we provide a guide on what factors to consider for designing functional metagenome-based studies on teleost microbiomes.
尽管针对硬骨鱼的微生物群研究激增，但很少有研究报告关于整个宏基因组的功能数据，因为从鱼类肠道样本中提取高生物量的微生物DNA已被证明很困难。斑马鱼是功能微生物群研究中一种很有前景的模式生物，但通过基于鸟枪法的宏基因组学对斑马鱼肠道微生物群的功能景观进行的研究仍然很少。因此，对于一种能够准确代表斑马鱼肠道微生物群或任何鱼类物种的合适采样方法，目前还缺乏共识。为了解决这个问题，我们系统地测试了四种斑马鱼肠道微生物群的采样方法：从鱼缸中收集粪便、整个肠道、肠道内容物，以及施加腹压以促进肠道物质的挤出。此外，我们还纳入了水样作为环境对照，以解决环境微生物群对每种样本类型的潜在影响。为了比较这些采样方法，我们采用了基因组解析宏基因组学和16S元条形码技术相结合的方法。我们在所有层面上观察到样本类型之间的差异，包括采样、生物信息处理、宏基因组共组装、宏基因组组装基因组（MAG）的生成、功能潜力、MAG覆盖率和群体水平的微多样性。与环境对照的比较突出了环境污染对数据解释的潜在影响。虽然所有测试的样本类型都能提供有关斑马鱼肠道微生物群的信息，但结果表明，研究鱼类微生物群的最佳样本类型取决于研究的具体目标，在这里我们提供了一份指南，介绍在设计基于宏基因组的硬骨鱼微生物群功能研究时应考虑哪些因素。
