Characterization of Shallow Whole-Metagenome Shotgun Sequencing as a High-Accuracy and Low-Cost Method by Complicated Mock Microbiomes.

Wenyi Xu,Hao Guo,Bowen Zhao,Xuesong Li,Jiaqi Yu,Fang Zhang,Yu Yang,Yanan Yang,Tianda Chen,Yuwei Pei,Zhuanyu Li,Chongming Wu

doi:10.3389/fmicb.2021.678319

Abstract

Characterization of the bacterial composition and functional repertoires of microbiome samples is the most common application of metagenomics. Although deep whole-metagenome shotgun sequencing (WMS) provides high taxonomic resolution, it is generally cost-prohibitive for large longitudinal investigations. Until now, 16S rRNA gene amplicon sequencing (16S) has been the most widely used approach and usually cooperates with WMS to achieve cost-efficiency. However, the accuracy of 16S results and its consistency with WMS data have not been fully elaborated, especially by complicated microbiomes with defined compositional information. Here, we constructed two complex artificial microbiomes, which comprised more than 60 human gut bacterial species with even or varied abundance. Utilizing real fecal samples and mock communities, we provided solid evidence demonstrating that 16S results were of poor consistency with WMS data, and its accuracy was not satisfactory. In contrast, shallow whole-metagenome shotgun sequencing (shallow WMS, S-WMS) with a sequencing depth of 1 Gb provided outputs that highly resembled WMS data at both genus and species levels and presented much higher accuracy taxonomic assignments and functional predictions than 16S, thereby representing a better and cost-efficient alternative to 16S for large-scale microbiome studies.

Highlights

The booming metagenomics enables direct cell-free analysis of microbial communities from environmental habitats and greatly benefits a vast range of fields, including human health, drug development, agriculture, and ecology
Getting the taxonomic profiles of the microbiome is a major concern for metagenomic investigations, which is mainly achieved through two sequencing approaches, 16S rRNA gene amplicon sequencing (16S) and deep whole-metagenome shotgun
Our findings demonstrate that 16S analysis is significantly inferior to S-wholemetagenome shotgun sequencing (WMS) in terms of consistency with WMS, recall rate, and accuracy at the genus level

Summary

Introduction

The booming metagenomics enables direct cell-free analysis of microbial communities from environmental habitats and greatly benefits a vast range of fields, including human health, drug development, agriculture, and ecology. WMS can provide taxa information down to species and even strain levels as well as analysis of functional potentials, and the high cost and massive data work leave it inaccessible to many researchers, ones performing large longitudinal studies. Researchers compromise to perform 16S sequencing at the beginning of studies to gain primary clues for the subsequent WMS studies. This strategy requires 16S results to be sufficiently accurate and highly consistent with the WMS datasheet. The accuracy and consistency of 16S readouts with WMS outputs have not been thoroughly investigated, mainly because there are few sophisticated artificial microbiomes with defined taxonomic information and similar complexity to the real microbial samples

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Discussion

Conclusion