PDF(Pubmed)
Abstract：
Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias.
摘要：
使用大小等级过滤对水生浮游生物（浮游细菌）群落进行多样性研究，DNA提取,系统发育标记的PCR和测序，需要一个强大的方法论管道，由于偏见基本上在各个层面都得到了证明，包括DNA提取,引物选择和PCR。即使相同浮游生物样品的过滤体积不同，因此，过滤器的不同生物质负载，会扭曲测序结果。在这项研究中,我们设计了一种基于Arduino微控制器的流量计，该流量计记录了初始（最大）流量的降低，作为浮游生物过滤过程中增加生物质负荷和过滤器堵塞的代理。该装置使用康斯坦茨湖的淡水浮游生物进行了测试，提取总DNA，并对16SrDNA扩增子进行测序。我们证实,用于相同水样品的不同过滤体积影响测序结果。在alpha和beta多样性以及所有分类学等级中都可见差异。受影响最大的类群是典型的淡水放线菌和拟杆菌，相对丰度增加到38%，减少到29%，分别。在另一个实验中,湖水样品未经稀释过滤，稀释三倍，并且一旦流量降低到初始流量的50%，就停止每次过滤，因此，在相同程度的过滤器堵塞。稀释三倍的样品需要三倍的过滤体积，同时提取了等量的总DNA，与未稀释的对照相比，所有分类等级的差异均无统计学意义。总之,这项工作证实了基于测序的群落分析的依赖于体积/生物量的浮游细菌过滤偏差，并提供了一种改进的程序，用于在过滤过程中控制生物量负荷，并独立于浮游生物密度从样品中回收等量的DNA。该装置的应用还可以避免由浮游生物过滤偏差引起的测序结果的失真。
