Department of Human Genetics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Institute of Food Technology, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Debrecen, Hungary.
Sci Rep. 2020 Feb 25;10(1):3419. doi: 10.1038/s41598-020-60304-y.
Here, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.
在这里，我们开发了一些方案来提高基于 16S rRNA 基因扩增的下一代测序（NGS）结果的灵敏度、严谨性和可比性。通过评估提取效率，包括产量、纯化 DNA 的纯度和片段化程度，以及考虑质量读数、扩增子序列变体（ASVs）、群落结构和生物多样性的测序指标，我们进行了一项全面的研究。我们确定了批次效应会显著影响肉鸡胃肠道（GIT）群落组成，并提出了改进灵敏度、一致性和跨研究可比性的建议。我们发现，提取核酸的纯度对下游文库制备的成功率有很强的影响。从粪便中制备粪便细菌悬浮液通过富集革兰氏阴性菌并成功地对低丰度类群进行编目，对群落生物多样性有显著的积极影响，比直接处理粪便材料的效果要好。依赖罗氏 MagNa Pure 24 磁珠自动化方法的应用提供了高度一致的结果，因此它似乎是研究肉鸡 GIT 微生物组的大规模研究中的最佳选择。
