# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
###### Abstract  
Cave microbiota knowledge has greatly expanded in the past decades, driven by the development of molecular techniques, which allow an in-depth characterization of diversity and its metabolic potential. This review focuses on the contribution of DNA sequencing technologies to depict the cave microbiome for the three domains of life (_Bacteria_, _Archaea_, and Microeukaryotes), assessing their advantages and limits. Cultural methods do not provide a representative view of cave microbial diversity but do offer, subsequently, the possibility to genomically characterize the strains isolated from caves. Next-generation DNA sequencing permits an exhaustive description of microbial biodiversity in caves, using metabarcoding (for taxonomic assessment) or metagenomics (for taxonomic and functional assessments). It proved useful to compare caves, different rooms, or substrata (water, soil, bedrock, etc.) within a cave, or the effect of cave disturbance in Lascaux and elsewhere. The integration of next-generation DNA sequencing with cultivation techniques, physico-chemical characterization, and other complementary approaches is important to understand the global functioning of caves and to provide key information to guide cave conservation strategies.  
microbial ecology; next-generation DNA sequencing; metabarcoding; metagenomics 20202  
processes that can occur on cave walls. For example, in many caves presenting famous paintings or engravings such as Lascaux Cave (France) or Altamira Cave (Spain), _Fungi_ and _Bacteria_ are reported as causing black or yellow stains [8; 11]. In Lascaux Cave, these colored stains were the result of fungal pigmentation, i.e., _Ochroconis lascauxensis_ (syn. _Sociocohashium lascauxense_) and _Ochroconis anomala_ synthesize black melanin pigments [12], while in Altamira Cave, the yellow pigmentation was attributed to _Betaproteobacteria_ and _Gammaproteobacteria_ activity [11]. While each cave is unique due to the particularity of its biological, chemical, and physical features [1; 5; 13], knowledge about microbial diversity, its interactions, and its impact on ecosystem functioning represents a challenge to address fundamental microbial ecology questions, but also conservation issues associated with caves.  
The number of cave microbiota studies has greatly expanded in the last decade, driven by large-scale conservation programs [14] and enabled by the development of high-resolution molecular techniques coupled with DNA Next-Generation Sequencing (NGS). Indeed, beyond classical microbial ecology methods relying on microbial cultivation, NGS techniques have promoted the emergence of new high-throughput sequencing-based approaches, such as metabarcoding, metagenomics, or metatranscriptomics [15], with the potential for (i) an in-depth characterization of microbial diversity in caves, (ii) the assessment of their genetic potential and metabolic capacities, and (iii) the possibility of focusing even on individual strains by using genomics or transcriptomics. It is now essential to consider complementary and multidisciplinary techniques to investigate microbial diversity thriving in caves, including culture-dependent and -independent methods, allowing scientists to gain insights into a global microbial functioning of caves and offering the possibility to guide conservation management politics.  
This review summarizes the contribution of the various technologies, from cultural methods to NGS sequencing, to our current knowledge on microbial diversity associated with cave ecosystems. A particular emphasis is put on molecular techniques integrating genomic reconstruction approaches as a perspective for non-cultivated microorganisms. Their relevance in interdisciplinary science to understand cave ecology is also discussed.