Van Syoc Emily, Carrillo Gaeta Natália, Ganda Erika
Integrative & Biomedical Physiology and Clinical & Translational Sciences Dual-Title Ph.D. Program, Pennsylvania State University, University Park, PA 16802, USA.
Department of Animal Science, College of Agricultural Sciences, Pennsylvania State University, University Park, PA 16801, USA.
Animals (Basel). 2021 May 12;11(5):1372. doi: 10.3390/ani11051372.
As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.
随着微生物组科学在农业中的应用不断扩展，应持续评估实验室方法，以确保下游结果的优化和可靠性。大多数动物微生物组研究使用粪便样本或直肠拭子来分析肠道细菌群落；然而，对于鸟类而言，由于泄殖腔独特的解剖结构，粪便、尿液和生殖道在一个孔口处汇合，因此难以采用上述方法。所以，禽类肠道微生物组通常从泄殖腔拭子中取样，这就需要评估样本制备方法以优化16S测序。我们比较了两种市售试剂盒中的四种不同DNA提取方法，这些方法用于从10只成年商品蛋鸡的泄殖腔拭子中提取DNA，同时设置了模拟群落和阴性对照，随后对其进行16S rRNA扩增子测序。评估了提取的DNA产量和质量、多样性分析以及污染物情况。观察到DNA质量和数量存在差异，并且所有方法都需要进一步纯化以实现最佳测序，这表明存在由于泄殖腔内容物、方法试剂和/或环境因素导致的污染物。然而，不同方法之间在α或β多样性方面未观察到差异。重要的是，在每个模拟群落和阴性对照中都检测到了多种细菌污染物，这表明实验室污染、操作污染以及方法特异性试剂污染普遍存在。我们发现，尽管提取方法导致了不同的提取质量和产量，但总体测序结果并未受到影响，并且我们没有发现任何一种方法在从泄殖腔拭子中提取DNA用于16S rRNA测序时是不合适的选择。总体而言，我们的结果强调了除DNA分离方法外，还需仔细考虑阳性和阴性对照的必要性，并为未来家禽微生物组研究提供了指导。
