Under-detection of endospore-forming Firmicutes in metagenomic data

Sevasti Filippidou,Thomas Junier,Tina Wunderlin,Chien-Chi Lo,Po-E Li,Patrick S Chain,Pilar Junier

doi:10.1016/j.csbj.2015.04.002

Abstract

Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methods of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group (spo0A and gpr). Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.

Highlights

Metagenomic studies have emerged as promising methods for the collective study of microbial communities directly extracted from environmental samples [1,2,3]
Our results indicate that endospore-forming Firmicutes are overlooked in environmental diversity surveys using traditional whole metagenomic approaches
In recent studies analyzing the minimal set of endospore-formation genes required by endosporeformers had indicated that spo0A is one of the most conserved genes almost exclusively found among this bacterial group [44,45,46]

Summary

Introduction

Metagenomic studies have emerged as promising methods for the collective study of microbial communities directly extracted from environmental samples [1,2,3] These approaches have been successfully applied to a variety of environments and have helped to unveil new functional pathways and metabolic processes within the microbial world [4,5,6,7,8]. Can occur at all the steps involved in a metagenomic workflow They can be associated to the specific type of environment [9, 10], the DNA yields obtained [11], the DNA extraction method [12], the amplification (for example in amplicon sequencing), and in the sequencing and the analysis of the sequences. In order to overcome these limitations, new approaches have been developed including single-cell genomics or culture-dependent methodologies such as culturomics [15,16] which, in their turn, have their own limitations

Objectives

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Results

Conclusion