State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Appl Microbiol Biotechnol. 2023 Jun;107(12):4109-4117. doi: 10.1007/s00253-023-12576-3. Epub 2023 May 16.
DNA-based analyses have become routine methods in soil microbial research, for their high throughput and resolution in characterizing microbial communities. Yet, concerns arise regarding the interference of relic DNA in estimates of viable bacterial community composition and individual taxa dynamics in soils that recovered from post-gamma irradiation. In this study, different soil samples with varying bacterial diversity but similar soil properties were randomly selected. We split each sample into two parts: one part was treated with propidium monoazide (PMA) before DNA extraction, PMA can bind to relic DNA and inhibit PCR amplification by chemical modification; DNA of the other part was extracted following the same process but without PMA pretreatment. Then, soil bacterial abundance was quantified by quantitative polymerase chain reaction, and bacterial community structure was examined by Illumina metabarcoding sequencing of 16S rRNA gene. The results showed that the higher bacterial richness and evenness were estimated when relic DNA was present. The variation trends of bacterial abundance, alpha diversity, and beta diversity remained the same, as reflected by the significant correlations between PMA-treated and -untreated samples (P < 0.05). Moreover, as the mean abundance increased, the reproducibility of detecting individual taxa dynamics between relic DNA present and absent treatments increased. These findings provide empirical evidence that a more even distribution of species abundance derived from relic DNA would result in the overestimation of richness in the total DNA pools and also have crucial implications for guiding proper application of high-throughput sequencing to estimate bacterial community diversity and taxonomic population dynamic. KEY POINTS: • Relic DNA effects on the bacterial community in sterilized soils were assessed. • More even species abundance distribution in relic DNA overestimates true richness. • The reproducibility of individual taxa dynamics increased with their abundance.
基于 DNA 的分析已成为土壤微生物研究中的常规方法，因为它们在表征微生物群落方面具有高通量和高分辨率的特点。然而，人们对放射性后土壤中可培养细菌群落组成和个体分类群动态的估计中残留 DNA 的干扰表示担忧。在这项研究中，随机选择了具有不同细菌多样性但具有相似土壤特性的不同土壤样本。我们将每个样本分为两部分：一部分在提取 DNA 之前用吖啶橙（PMA）处理，PMA 可以与残留 DNA 结合，并通过化学修饰抑制 PCR 扩增；另一部分的 DNA 按照相同的过程提取，但不进行 PMA 预处理。然后，通过定量聚合酶链反应定量土壤细菌丰度，并通过 Illumina 16S rRNA 基因代谢组测序检测细菌群落结构。结果表明，当存在残留 DNA 时，估计的细菌丰富度和均匀度更高。细菌丰度、α多样性和β多样性的变化趋势相同，这反映了 PMA 处理和未处理样品之间存在显著相关性（P < 0.05）。此外，随着平均丰度的增加，在存在和不存在残留 DNA 处理的情况下检测个体分类群动态的重现性增加。这些发现为以下观点提供了经验证据，即来自残留 DNA 的物种丰度更均匀的分布将导致总 DNA 池中丰富度的高估，并且对指导高通量测序正确应用以估计细菌群落多样性和分类群种群动态具有重要意义。关键点：• 评估了灭菌土壤中细菌群落的残留 DNA 影响。• 残留 DNA 中更均匀的物种丰度分布高估了真实的丰富度。• 个体分类群动态的重现性随着其丰度的增加而增加。
