Lanzén Anders, Lekang Katrine, Jonassen Inge, Thompson Eric M, Troedsson Christofer
NEIKER-Tecnalia, Department of Conservation of Natural Resources, Bizkaia Technology Park, Derio, Spain.
Department of Biology, University of Bergen, Bergen, Norway.
PLoS One. 2017 Jun 16;12(6):e0179443. doi: 10.1371/journal.pone.0179443. eCollection 2017.
Human impact on marine benthic communities has traditionally been assessed using visible morphological traits and has focused on the macrobenthos, whereas the ecologically important organisms of the meio- and microbenthos have received less attention. DNA metabarcoding offers an alternative to this approach and enables a larger fraction of the biodiversity in marine sediments to be monitored in a cost-efficient manner. Although this methodology remains poorly standardised and challenged by biases inherent to rRNA copy number variation, DNA extraction, PCR, and limitations related to taxonomic identification, it has been shown to be semi-quantitative and useful for comparing taxon abundances between samples. Here, we evaluate the effect of replicating genomic DNA extraction in order to counteract small scale spatial heterogeneity and improve diversity and community structure estimates in metabarcoding-based monitoring. For this purpose, we used ten technical replicates from three different marine sediment samples. The effect of sequence depth was also assessed, and in silico pooling of DNA extraction replicates carried out in order to maintain the number of reads constant. Our analyses demonstrated that both sequencing depth and DNA extraction replicates could improve diversity estimates as well as the ability to separate samples with different characteristics. We could not identify a "sufficient" replicate number or sequence depth, where further improvements had a less significant effect. Based on these results, we consider replication an attractive alternative to directly increasing the amount of sample used for DNA extraction and strongly recommend it for future metabarcoding studies and routine assessments of sediment biodiversity.
传统上，人类对海洋底栖生物群落的影响是通过可见的形态特征来评估的，并且主要关注大型底栖生物，而生态上重要的小型底栖生物和微型底栖生物受到的关注较少。DNA宏条形码技术为这种方法提供了一种替代方案，能够以经济高效的方式监测海洋沉积物中更大比例的生物多样性。尽管这种方法的标准化程度仍然很低，并且受到rRNA拷贝数变异、DNA提取、PCR以及分类鉴定相关局限性所固有的偏差的挑战，但它已被证明是半定量的，并且有助于比较不同样本间的分类单元丰度。在此，我们评估复制基因组DNA提取的效果，以抵消小规模的空间异质性，并改善基于宏条形码技术监测中的多样性和群落结构估计。为此，我们使用了来自三个不同海洋沉积物样本的十个技术重复样本。我们还评估了测序深度的影响，并对DNA提取重复样本进行了虚拟合并，以保持读数数量恒定。我们的分析表明，测序深度和DNA提取重复样本都可以改善多样性估计以及区分具有不同特征样本的能力。我们无法确定一个“足够”的重复样本数量或测序深度，超过这个数量后进一步的改进效果就不太显著了。基于这些结果，我们认为复制是直接增加用于DNA提取的样本量的一种有吸引力的替代方法，并强烈建议在未来的宏条形码技术研究和沉积物生物多样性的常规评估中采用。
