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Sample Processing Methods Impacts on Rumen Microbiome

机译：样品处理方法对瘤胃微生物组的影响

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The standardization of collection and processing methods for rumen samples is crucial to reduce the level of errors that may affect the analysis and interpretation of the data. The aim of this study was to compare two processing methods and their impacts on the microbial community composition analysis, from material that was either immediately frozen or samples that were stored as cell pellets after removing the supernatant prior to freezing. Eight rumen-fistulated Brahman steers received chloroform as an antimethanogenic compound for 21 days. Rumen fluid samples (60 mL per animal) were collected using a probe covered with two layers of cheesecloth at 3 h post feeding at day 0 prior-treatment (control period) and day 21 of treatment. One sub-set of samples were placed in dry ice and stored at −80°C (Method 1) for subsequent DNA extraction, while a second subset of samples was centrifuged, the supernatant removed and the microbial pellet and rumen contents placed in dry ice and stored at −80°C (Method 2) prior to DNA extractions. Phylogenetic based methods (Illumina Miseq) targeting the 16S rRNA gene were used to characterize the bacterial and archaeal communities from both collection methods for the control and treatment periods. The results from this study showed that the chloroform treatment was significantly different for all beta diversity measures regardless of the processing method used. Significant differences in the relative abundances of some bacteria and archaea, such as Elusimicrobia, Fibrobacteres, Lentisphaerae, Spirochaetes, and Verrucomicrobia and Methanomassiliicoccaceae, were observed at higher levels in the Method 2. These microbial populations are known to have fragile cell wall structures and are susceptible to cell lysis. Regardless of the processing method used, both identified the key microbial groups and can be used to compare the relative shifts in the rumen microbiome between treatments. However, immediately freezing samples might alter the abundance of material from species that are more readily lysed and will not be suitable for studies that aim to assign absolute abundance values to these species within the rumen.
机译：瘤胃样品的收集和处理方法的标准化对于减少可能影响数据分析和解释的错误水平至关重要。这项研究的目的是比较两种处理方法及其对微生物群落组成分析的影响，这些方法既可以是立即冷冻的材料，也可以是在冷冻前去除上清液后以细胞沉淀形式存储的样品。八头瘤胃-裂的婆罗门ste牛以氯仿作为致死激素化合物接受了21天。在治疗前第0天（对照期）和治疗第21天，在喂食后3小时，使用覆盖有两层粗棉布的探针收集瘤胃液样品（每只动物60mL）。将一小组样品置于干冰中，并保存在-80°C（方法1）中，用于随后的DNA提取，同时将第二小组样品离心，除去上清液，并将微生物沉淀和瘤胃内容物置于干冰中并在提取DNA之前保存在-80°C（方法2）中。针对16S rRNA基因的基于系统发育的方法（Illumina Miseq）被用于表征对照和治疗期两种收集方法的细菌和古细菌群落。这项研究的结果表明，无论使用哪种加工方法，所有β多样性措施的氯仿处理均存在显着差异。在方法2中，观察到较高水平的某些细菌和古细菌（例如Elusimicrobia，Fibrobacteres，Lentsphaerae，Spirochaetes和Verrucomicrobia和Methanomassiliicoccaceae）的相对丰度存在显着差异。已知这些微生物种群具有脆弱的细胞壁结构，并且易于细胞裂解。无论使用哪种处理方法，都可以确定关键的微生物组，并且可以用来比较处理之间瘤胃微生物组的相对变化。但是，立即冷冻的样品可能会改变更容易裂解的物种中物质的丰度，因此不适合旨在为瘤胃内的这些物种指定绝对丰度值的研究。

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