Sequencing 16S rRNA gene amplicons is the gold standard to uncover the composition of prokaryotic communities. The presence of multiple copies of this gene makes the community abundance data distorted and gene copy normalization (GCN) necessary for correction. Even though GCN of 16S data provided a picture closer to the metagenome before, it should also be compared with communities of known composition due to the fact that library preparation is prone to methodological biases. Here, we process 16S rRNA gene amplicon data from eleven simple mock communities with DADA2 and estimate the impact of GCN. In all cases, the mock community composition derived from the 16S sequencing differs from those expected, and GCN fails to improve the classification for most of the analysed communities. Our approach provides empirical evidence that GCN does not improve the 16S target sequencing analyses in real scenarios. We therefore question the use of GCN for metataxonomic surveys until a more comprehensive catalogue of copy numbers becomes available.

测序16S rRNA基因扩增子是发现原核生物组成的金标准。该基因的多个拷贝的存在使社区的丰度数据失真，并且校正所需的基因拷贝归一化（GCN）。尽管16S数据的GCN之前提供的图像更接近元基因组，但由于文库制备容易受到方法学偏见的事实，也应将其与已知组成的群落进行比较。在这里，我们使用DADA2处理来自11个简单模拟社区的16S rRNA基因扩增子数据，并评估GCN的影响。在所有情况下，源自16S测序的模拟社区组成均与预期的有所不同，并且GCN无法改善大多数分析社区的分类。我们的方法提供了经验证据，表明GCN不能在实际场景中改善16S目标序列分析。因此，我们对在分类学研究中使用GCN表示怀疑，直到可以使用更全面的副本编号目录为止。