Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, 6220 MSRB III/SPC 5642, 1150 W. Medical Center Dr, Ann Arbor, MI, 48109-5642, USA.
Microbiome. 2021 May 5;9(1):99. doi: 10.1186/s40168-021-01055-4.
Low-biomass microbiome studies (such as those of the lungs, placenta, and skin) are vulnerable to contamination and sequencing stochasticity, which obscure legitimate microbial signal. While human lung microbiome studies have rigorously identified sampling strategies that reliably capture microbial signal from these low-biomass microbial communities, the optimal sampling strategy for characterizing murine lung microbiota has not been empirically determined. Performing accurate, reliable characterization of murine lung microbiota and distinguishing true microbial signal from noise in these samples will be critical for further mechanistic microbiome studies in mice.
Using an analytic approach grounded in microbial ecology, we compared bacterial DNA from the lungs of healthy adult mice collected via two common sampling approaches: homogenized whole lung tissue and bronchoalveolar lavage (BAL) fluid. We quantified bacterial DNA using droplet digital PCR, characterized bacterial communities using 16S rRNA gene sequencing, and systematically assessed the quantity and identity of bacterial DNA in both specimen types. We compared bacteria detected in lung specimens to each other and to potential source communities: negative (background) control specimens and paired oral samples. By all measures, whole lung tissue in mice contained greater bacterial signal and less evidence of contamination than did BAL fluid. Relative to BAL fluid, whole lung tissue exhibited a greater quantity of bacterial DNA, distinct community composition, decreased sample-to-sample variation, and greater biological plausibility when compared to potential source communities. In contrast, bacteria detected in BAL fluid were minimally different from those of procedural, reagent, and sequencing controls.
An ecology-based analytical approach discriminates signal from noise in this low-biomass microbiome study and identifies whole lung tissue as the preferred specimen type for murine lung microbiome studies. Sequencing, analysis, and reporting of potential source communities, including negative control specimens and contiguous biological sites, are crucial for biological interpretation of low-biomass microbiome studies, independent of specimen type. Video abstract.
低生物量微生物组研究（如肺部、胎盘和皮肤）容易受到污染和测序随机性的影响，从而掩盖了合法的微生物信号。虽然人类肺部微生物组研究已经严格确定了可靠地从这些低生物量微生物群落中捕获微生物信号的采样策略，但尚未通过实验确定用于描述鼠肺部微生物组的最佳采样策略。准确、可靠地描述鼠肺部微生物组，并区分这些样本中的真实微生物信号与噪声，对于进一步开展小鼠的机制微生物组研究至关重要。
我们使用基于微生物生态学的分析方法，比较了通过两种常见采样方法（肺匀浆和支气管肺泡灌洗（BAL）液）收集的健康成年小鼠肺部的细菌 DNA。我们使用液滴数字 PCR 定量细菌 DNA，使用 16S rRNA 基因测序描述细菌群落，并系统地评估了两种标本类型中细菌 DNA 的数量和身份。我们将肺部标本中检测到的细菌相互比较，并与潜在的源群进行比较：阴性（背景）对照标本和配对的口腔样本。所有指标均表明，与 BAL 液相比，小鼠的肺组织包含更多的细菌信号，污染证据更少。与 BAL 液相比，与潜在源群相比，肺组织具有更高的细菌 DNA 量、独特的群落组成、样本间变异性降低和更高的生物学合理性。相比之下，BAL 液中检测到的细菌与处理、试剂和测序对照的细菌几乎没有差异。
基于生态学的分析方法可在这项低生物量微生物组研究中区分信号和噪声，并确定肺组织是研究鼠肺部微生物组的首选标本类型。对潜在源群（包括阴性对照标本和相邻生物部位）的测序、分析和报告对于低生物量微生物组研究的生物学解释至关重要，与标本类型无关。视频摘要。
