PDF(Pubmed)
Abstract：
Molecular profiling techniques such as metagenomics, metatranscriptomics or metabolomics offer important insights into the functional diversity of the microbiome. In contrast, 16S rRNA gene sequencing, a widespread and cost-effective technique to measure microbial diversity, only allows for indirect estimation of microbial function. To mitigate this, tools such as PICRUSt2, Tax4Fun2, PanFP and MetGEM infer functional profiles from 16S rRNA gene sequencing data using different algorithms. Prior studies have cast doubts on the quality of these predictions, motivating us to systematically evaluate these tools using matched 16S rRNA gene sequencing, metagenomic datasets, and simulated data. Our contribution is threefold: (i) using simulated data, we investigate if technical biases could explain the discordance between inferred and expected results; (ii) considering human cohorts for type two diabetes, colorectal cancer and obesity, we test if health-related differential abundance measures of functional categories are concordant between 16S rRNA gene-inferred and metagenome-derived profiles and; (iii) since 16S rRNA gene copy number is an important confounder in functional profiles inference, we investigate if a customised copy number normalisation with the rrnDB database could improve the results. Our results show that 16S rRNA gene-based functional inference tools generally do not have the necessary sensitivity to delineate health-related functional changes in the microbiome and should thus be used with care. Furthermore, we outline important differences in the individual tools tested and offer recommendations for tool selection.
摘要：
分子谱分析技术，如宏基因组学，代谢组学或代谢组学为微生物组的功能多样性提供了重要的见解。相比之下,16SrRNA基因测序，一种广泛且具有成本效益的测量微生物多样性的技术，只允许间接估计微生物的功能。为了缓解这种情况，PICRUSt2，Tax4Fun2，PanFP和MetGEM等工具使用不同的算法从16SrRNA基因测序数据推断功能概况。先前的研究对这些预测的质量产生了怀疑，激励我们使用匹配的16SrRNA基因测序系统评估这些工具，宏基因组数据集，和模拟数据。我们的贡献有三个方面：(I)使用模拟数据，我们调查技术偏差是否可以解释推断和预期结果之间的不一致;（ii）考虑人类队列2型糖尿病，结直肠癌和肥胖症,我们测试功能类别的健康相关差异丰度测量是否在16SrRNA基因推断和宏基因组来源的谱之间一致;（iii）由于16SrRNA基因拷贝数是功能谱推断中的重要混淆者，我们调查使用rrnDB数据库定制的拷贝数标准化是否可以改善结果.我们的结果表明，基于16SrRNA基因的功能推断工具通常没有必要的敏感性来描绘微生物组中与健康相关的功能变化，因此应谨慎使用。此外，我们概述了所测试的各个工具的重要差异，并为工具选择提供了建议。
