Profiling the 16S-rRNA gene is a common method in microbial ecology to provide estimates of microbial diversity and abundance, but it cannot directly produce information about the functional potential of these taxa. While shotgun sequencing can provide functional information, the cost can be prohibitive for large scale comparative studies. Functional prediction tools have been developed to generate expected gene counts from 16S-rRNA amplicon data and allow for initial exploration of hypotheses prior to shotgun sequencing. PICRUSt2 and Tax4Fun2 are two such prediction tools that are becoming increasingly popular in microbial ecology studies. However, there are only a few benchmarking studies that have tested their performance, and none that focused on soils. The goal of the present study was to compare the performance of both prediction tools with each other and shotgun sequencing. We assessed prediction output from both tools used on the same samples from two sites in Southwest Florida and compared the predicted functional profiles with MG-RAST functional profiles on a shotgun-sequenced subset of samples. Shotgun and amplicon sequencing datasets showed comparable 16S rRNA gene profiles with observed differences likely stemming from PCR bias and taxa-dependent variability of 16S rRNA gene density in shotgun sequences. PICRUSt2 functional profiles represented greater relative abundances of genes in the pathway for oxidative phosphorylation, while Tax4Fun2 detected more genes from specialized metabolic pathways, such as for methane metabolism. PICRUSt2 functional profiles showed somewhat stronger correlations with shotgun-sequenced functional profiles than Tax4Fun2. However, both prediction tools significantly underestimated gene frequencies in many KEGG categories, including genes with biogeochemical significance for soil carbon- and nitrogen cycling.

分析 16S-rRNA 基因是微生物生态学中提供微生物多样性和丰度估计值的常用方法，但它不能直接产生有关这些分类群功能潜力的信息。虽然霰弹枪测序可以提供功能信息，但大规模比较研究的成本可能过高。已经开发出功能预测工具来从 16S-rRNA 扩增子数据生成预期的基因计数，并允许在鸟枪法测序之前对假设进行初步探索。PICRUSt2 和 Tax4Fun2 是两个这样的预测工具，它们在微生物生态学研究中越来越受欢迎。然而，只有少数基准研究测试了它们的性能，而且没有一个专注于土壤。本研究的目的是比较这两种预测工具的性能和鸟枪法测序的性能。我们评估了在佛罗里达州西南部两个地点的相同样本上使用的两种工具的预测输出，并将预测的功能配置文件与霰弹枪测序的样本子集上的 MG-RAST 功能配置文件进行了比较。鸟枪法和扩增子测序数据集显示出相似的 16S rRNA 基因谱，观察到的差异可能源于 PCR 偏差和鸟枪法序列中 16S rRNA 基因密度的分类群依赖性变异性。PICRUSt2 功能谱代表氧化磷酸化途径中基因的相对丰度更高，而 Tax4Fun2 检测到更多来自专门代谢途径的基因，例如甲烷代谢。与 Tax4Fun2 相比，PICRUSt2 功能配置文件与霰弹枪测序功能配置文件显示出更强的相关性。然而，这两种预测工具都显着低估了许多 KEGG 类别中的基因频率，包括对土壤碳和氮循环具有生物地球化学意义的基因。