2.3 Molecular resolution

The contents of this section have been extracted and modified from the article Disentangling host–microbiota complexity through hologenomics published in Nature Reviews Genetics in 2022 by the authors of the Holo-omics Workbook.

The complexity of a study system is not only determined by its inherent properties and study design, but also the techniques and procedures employed to analyse it. Researchers can decide how much a system is simplified by altering the resolution of the hologenomic features under study; in essence, zooming in or zooming out.

2.3.1 Resolution of host genotypes

In host-microbiota studies, host genotypes can be defined at different levels, including species, breeds, populations, strains, sex or individuals. Genotypes can be defined as categorical variables, without analysing the differences between them, or can be studied in more detail through considering their actual genetic content and establishing correlations among them. When using an evolutionary perspective, phylogenetic relationships between genotypes are established based on phylogenomic markers, which usually vary above population and species level, but not among individuals. This implies that genomic variability among the individuals included within each genotype is overlooked. Studying the effect of interindividual genomic variability on host-microbiota systems, such as identifying candidate host genomic variants associated with microbial features, requires a higher level of resolution. This is achieved through defining genotypes at the individual level, and using techniques based on whole genome resequencing that enable the complexity of host genomes to be screened at a much finer level, so that differences between the individuals contrasted are not only defined based on their kinship, but also the functional properties of their genomic variants. Currently, this approach requires high quality reference genomes from which high density SNP profiles of individuals can be generated, for example through SNPchip or resequencing studies. The genomic resolution could be further refined by incorporating structural variants, methylation patterns, or even, we hypothesise, chromosome 3D folding structure as revealed through techniques such as Hi-C. In doing so, researchers can identify associations between SNPs or gene variants and specific microbiota traits, such as the relative abundance of certain taxa or the enrichment of a given function, and thus identify mechanisms by which a host exerts control over composition and function of its associated microbiota

Resolution of microbial metagenotypes

The structure and resolution at which microbial metagenotypes are defined also affects the complexity of the metagenome under analysis. Metagenotypes can be defined as arrays of microbial taxa, microbial genes or a combination of both. The most common approach to define them is to rely on short marker sequences targeted for metabarcoding purposes, such as the 16S rRNA or the internal transcribed spacer (ITS). However, these procedures often do not enable reliable taxonomic assignment at genus or species level, do not capture strain level community dynamics, and are prone to generate biased functional inferences, as bacteria with identical marker genes (particularly those associated with wild taxa) might carry very different catalogues of genes. Thus, while useful for estimating microbial diversity and obtaining preliminary insights into functionality, targeted sequencing approaches do not provide conclusive evidence about the metabolic capabilities of the microbiota, particularly when working with non-human systems.

By contrast, if appropriate strategies and adequate sequencing depths are employed, shotgun metagenomics enables bacterial genome sequences to be recovered, from which genes can be predicted and annotated to create a gene catalogue that can define a metagenotype. However, these genes are not randomly distributed, but enclosed within genomes of specific bacteria or other microorganisms, with a particular combination of genes that shape their expression and the specific biological features (such as oxygen affinity, reproduction time, metabolic capacity) that determine their ecology. Hence, a more refined characterisation of microbial metagenotypes can be achieved through binning algorithms that enable bacterial genome reconstruction from metagenomic mixtures, yielding metagenome-assembled genomes (MAGs). Nevertheless, unless short-read sequencing is combined with long-read approaches, it is challenging to capture multi-copy genes such as the 16S rRNA marker gene 103, which is often employed in metabarcoding studies and therefore represents a useful link to a large number of existing studies. Machine learning-based solutions to link 16S rRNA marker gene sequences with MAGs are, however, being developed 104. Finally, regardless of the approach used to define the microbial metagenotype, the complexity of microbial communities will often require dimensionality reduction to increase statistical power 105,106. This can be achieved by defining co-abundance clusters, ecological guilds or more complex strategies that also consider temporal features of microbiota variation, such as compositional tensor factorisation.

Resolution of envirotypes

Characterisation of environmental factors that affect the host-microbiota system under study enable the definition of envirotypes, a term drawn from crop sciences that is useful for accounting for the environmental factors in the hologenomic context. Any different physical place, or place sampled at different time points, will be exposed to a different environment, as conditions will seldom be identical between two spatial and temporal points. Hence, the resolution at which the composite of environmental factors is considered will define whether these two environments will be considered different envirotypes or not. For example, if only considering water temperature, killer whales sampled in the Arctic and the Antarctic seas experience the same envirotype. However, if the biotic composition is also considered in the definition of the environment, the Arctic and the Antarctic will need to be split into two distinct envirotypes, as some killer whales will have access to penguins while others will not. The same principle applies to laboratory setups or mesocosm experiments: a temperature shift of 2-3 ºC might not be considered relevant under some experimental setups, while it can define different envirotypes under other study designs. Finally, failure to recognise environmental factors that affect host-microbiota interactions, and thus define relevant envirotypes, can lead to increased noise and decreased capacity to achieve statistical significance.