Ibarbalz Federico M, Pérez María Victoria, Figuerola Eva L M, Erijman Leonardo
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina; Agua y Saneamientos Argentinos S.A. (AySA), Buenos Aires, Argentina.
PLoS One. 2014 Jun 12;9(6):e99722. doi: 10.1371/journal.pone.0099722. eCollection 2014.
The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point.
比较了两组靶向16S rRNA基因V1-V3和V4可变区的引物在描述全尺寸活性污泥中细菌多样性变化和时间周转方面的性能。尽管两个16S rRNA区域的高通量扩增子测序数据的重复样本共享了一系列十二个月度样本中观察到的核心分类群集合，但每个分类群的相对丰度在不同区域之间存在显著差异。一个恰当的例子是丝状细菌硫丝菌相对丰度的变化，它对多样性指数有很大影响，但仅在V1-V3数据集中。然而，来自两个区域的扩增子测序数据中硫丝菌的相对丰度与使用荧光原位杂交确定的其丰度估计值相关。在非度量多维分析中，样本是根据测序区域而不是样本身份沿着第一排序轴分布的。微生物群落动态表明，16S rRNA基因的V1-V3和V4区域产生了可比的模式：1）群落内沿固定时间间隔发生的变化；2）活性污泥群落的缓慢周转；3）根据分类群-时间关系计算的物种替代率。温度是唯一与使用两对引物获得的数据集随时间的细菌群落组成显示出显著相关性的操作变量。总之，我们表明，尽管扩增子测序会引入偏差，但V1-V3和V4可变区可以可靠地用于细菌群落动态的定量评估，并对群落中的一般分类群提供适当的定性描述，特别是当数据是在一个方便的时间窗口内而不是在单个时间点获得时。
