Abstract
Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites).
Highlights
Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities
We previously introduced a profiling tool that uses universally occurring, protein coding, single copy phylogenetic marker gene (MG)-based operational taxonomic units as an approach to capture and quantify microbial taxa at species-level resolution in metagenomic samples9. MG-based operational taxonomic units (mOTUs) are built on the basis of MGs from both known and unknown species, the latter of which are extracted from existing metagenomes, enabling higher taxonomic resolution and more accurate quantification of species when profiling new microbial communities[9]
To obtain species-level taxonomic groups of sequences, we clustered these genomes based on a calibrated cutoff of 96.5% sequence identity[4] into 5232 non-redundant, reference MG-based operational taxonomic units that contained more than half of a subset of ten MGs that were found suitable for metagenomic analyses[9]
Summary
Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. The most common approach for microbial community profiling is by classification of PCR amplicon sequences from the small subunit ribosomal RNA gene (i.e., the 16S rRNA gene of bacteria and archaea) While powerful, this approach is known to introduce biases in composition estimates due to, for instance, variations in 16S rRNA gene copy numbers per genome (Supplementary Figure 1), unequal efficiencies of PCR-primers in different species[1, 2] as well as the use of different sub-regions of this gene[3]. These include organisms—hereon referred to as ‘unknown’ species— that may be detected, but remain difficult to quantify using standard methods and up-to-date genome databases To overcome this issue, we previously introduced a profiling tool that uses universally occurring, protein coding, single copy phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) as an approach to capture and quantify microbial taxa at species-level resolution in metagenomic samples. We previously introduced a profiling tool that uses universally occurring, protein coding, single copy phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) as an approach to capture and quantify microbial taxa at species-level resolution in metagenomic samples9. mOTUs are built on the basis of MGs from both known and unknown species, the latter of which are extracted from existing metagenomes, enabling higher taxonomic resolution and more accurate quantification of species when profiling new microbial communities[9]
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