# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 5 Taxonomic and Functional Assessment of Cave Microorganisms by Metagenomics
### Comparison between Metabarcoding and Metagenomic Approaches in Cave Ecosystems  
Few studies have compared metabarcoding and metagenomic approaches to document microbial diversity, and even fewer in caves. However, the compilation of various studies presented in Figure 1 allows us to establish a comparison between these two methods for the three domains of life in different cave compartments. Four literature comparisons can be made for the bacterial community (water and rock walls for anthropized caves and sediments and speleothems for non-anthropized caves), one for the archaeal community (rock walls for anthropized caves), and one for the microeukaryotic community (sediment for non-anthropized caves). Whatever the comparison, differences in community structure and composition were found. For example with the sediments of non-anthropized caves, the relative abundance of the _Basidiumyota_ microeukaryotic phylum was higher with the metagenomic approach, where it represented \(>\)5% of sequences (Figure 1 and references therein). In speleothems, 4 bacterial phyla were detected by metabarcoding versus 16 using metagenomics, with a 35% decrease in the relative abundance of the _Firmicutes_ phylum when considering metagenomic data compared with metabarcoding. Another comparison in the sediments of Manao-Pee Cave identified 123 bacterial families by metagenomicsagainst 55 families with the 16S rRNA dataset [103; 125]. For the archaeal community, the proportion of sequences affiliated to the _Thuamarchaeota_ and _Euryarchaeota_ phyla was also very different between the two approaches. In Weebubble Cave (Australia), the relative abundance of _Archaea_ was \(\sim\)40% in the 16S rRNA amplicon dataset versus only \(\sim\)20% in the binned metagenomic dataset [117]. The different copy numbers of the 16S and 18S rRNA genes in microbial genomes can lead to contrasted results. Since the number of 16S rRNA gene copies per genome for each single species or strain present in microbial assemblages is not predictable, consequently no fully effective corrections in metabarcoding and/or metagenomic datasets can be implemented to better reflect environmental reality.  
Metabarcoding and metagenomic approaches display different advantages and drawbacks to characterize taxonomic diversity. On the one hand, metabarcoding is a powerful tool for exhaustively describing the diversity of _Archaea_, _Bacteria_, and _Eukaryota_ in caves and other ecosystems. In addition, metabarcoding sequencing is less expensive than metagenomics. On the other hand, metabarcoding does not allow quantitative comparisons when different markers are used. In addition, metabarcoding is PCR based, whereas DNA is directly fragmented and sequenced in metagenomics, without any PCR bias. Finally, metagenomics has the advantage of assembling large fragments, sometimes entire reconstructed genomes of high quality (in terms of contamination and coverage), allowing scientists to discriminate microorganisms at the species or even strain level. This is not possible with the short amplicons used for metabarcoding, whose resolution is generally limited to the genus level [56; 103]. Therefore, metagenomics is more appealing to uncover the contribution of each microbial domain to the microbial assemblage in caves.  
In addition to taxonomic information, shotgun metagenomics also provides information on the genetic potential of microorganisms, allowing a better understanding of microbial adaptation and functional capacities in the environment investigated. The functional diversity encoded in the metagenome can only approximate the functionality of the community, as the presence of a gene does not mean that it is expressed at the time of collection. Analysis of metatranscriptomic and metaproteomic data can provide an additional insight into pathways that are actively expressed in the community, but this remains to be implemented in caves.