Scientists from Aachen set standards for microbiome analysis by metagenomics

Scientists at Uniklinik RWTH Aachen have benchmarked a key method used to characterize the microbial contents of our digestive tracts. Their work will enable scientists to more clearly understand which bacteria inhabit our guts and how they influence our health.

Humans harbour trillions of microbes, mostly in our guts, that exert important effects on many aspects of human biology, from digestion to immunity and even mental health. Thus, understanding which microbes reside in our guts and how they function is an important goal. Now, researchers led by Univ.-Prof. Dr. Thomas Clavel, Professor for Intestinal Microbiology at Uniklinik RWTH Aachen, have rigorously assessed the potentials and limits of a key method, metagenomic sequencing, used to answer these questions. Their study, which was carried out in collaboration with colleagues from Vienna (Austria) and Maastricht (Netherlands), is entitled “Benchmarking of shotgun sequencing depth reveals the potential and limitations of shallow metagenomics and strain-level analysis” and is published in the journal Nature Microbiology. 

Scientists typically study microbiomes by determining the DNA sequences of the microbes in the community, which tells them what microbes are present and what genes they harbour. Prof. Clavel, first author Dr. Nicole Treichel, and colleagues focused their attention on an approach for DNA sequencing called shotgun sequencing, a method in which DNA molecules are randomly broken into small fragments, sequenced individually, and then reassembled into overlapping fragments using bioinformatics. Even though it is widely used to study bacterial communities, the approach had so far not been thoroughly tested using reference datasets of known composition.

The team started by constructing different complex mixtures of bacterial DNA with varying distributions of different bacteria. Sequencing these mixtures where the composition of microbes was known allowed the researchers to determine what information can be gleaned at what sequencing depth. Importantly, determining the taxonomic diversity of a complex community is shown to be possible even at a comparatively shallow sequencing depth (<1 GB per sample) when a reference genome database is available, an important consideration due to the costs of deeper sequencing in large-scale studies. However, reconstructing the entire genetic material of single bacterial strains, also referred to as metagenome-assembled genomes (MAGs), requires a sequencing depth that is 10 times higher than what is needed to determine the taxonomic diversity, and this process is also prone to generating artefacts. The researchers’ analysis also showed what sequencing depths are required to characterize the functions of microbiome members, with a 5-fold higher depth required to identify individual proteins compared to simply describing biological processes. Being able to compare results between different laboratories is crucial. The authors found that while different protocols and high amounts of background DNA may confound results, this can be mitigated by sequencing samples at higher depth.

Prof. Clavel: “Diversity between strains contributes to functional differences in microbiomes between individuals. So, there is great interest in reconstructing the genomes of individual microbiome members. However, based on our results we can conclude that reconstructing MAGs is a complicated endeavour, even at high sequencing depths. In fact, only 50–80% of these sequences are correct, depending on the bioinformatic approach used.”

The researchers’ work was carried out as part of two projects, SFB1382 “Gut-liver-axis” and NFDI4Microbiota, funded by the German Research Foundation (DFG). 

https://doi.org/10.1038/s41564-026-02334-2