Under-detection of endospore-forming Firmicutes in metagenomic data

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.

Keywords: Endospores; Metagenomics; Profile analysis; gpr; spo0A.

Fig. 1

A. Validation of the profiles created for the genes spo0A and gpr compared to a selection of genomes of endospore-forming Firmicutes (blue bars) and non spore-forming genomes (red bars). In endospore-forming Firmicutes a single hit with a score above 2000 (Spo0A) and 2500 (Gpr) distinguish between positive and negative hits. Strco = Streptomyces coelicolor; Rhime = Rhizobium melliloti; Nosaz: Nostoc azollae; Lacac = Lactobacillus acidophilus; Escco = Escherichia coli; Desre = Desulfotomaculum reducens; Desha = Desulfitobacterium hafniense; Clobo = Clostridium botulinum; Bacha = Bacillus halodurans; Aliac = Alicyclobacillus acidocaldarius. B. The same analysis was repeated using all 59 endospore-forming genomes retrieved from IMG and CMR databases (see supplementary Table 1).

Fig. 2

Profile similarity hits for Spo0A and Gpr protein profiles in metagenomes from different origins. The color code identifying different environments is presented under the results. The genomes included in profile testing (see Fig. 1A) were also included in the analysis and are presented in white (endospore-formers) and gray (non-spore formers).

Fig. 3

Analysis of pyrosequencing results obtained from 16S rRNA gene and spo0A amplicons, from an environmental sample with high prevalence of endospore-forming Firmicutes (Nea Apollonia, NAP). (A) Total 16S rRNA gene community composition to the phylum level. (B) Firmicute fraction of the total community (16S rRNA gene) to the genus level. (C). Cladogram representing the community composition of Firmicutes using the spo0A gene. Sequences color coded by genus.