About 95% of global liquid freshwater is stored in the terrestrial subsurface, making it the largest terrestrial freshwater biome. Despite the importance of this water source for humanity, these systems are largely understudied from the microbiological point of view. In Slovenia, about half of the population depends on groundwater for its domestic needs. The quality of this water source and the putative presence of pathogens are not properly monitored. Accordingly, very little is known about the composition and the structure of its native prokaryotic communities. Here presented project aims to lay ground for microbiological and ecological studies of groundwater reservoirs of Slovenian karst. It aims to identify and describe the diversity in those pristine reservoirs in which the microbiota is still present in its original splendor. To achieve this, we plan to take advantage of recently emerged approaches in metagenomics. In more detail, we plan to analyze a timeseries of short insert and fosmid libraries constructed in parallel from environmental DNA extracted from pristine groundwater reservoirs. Prior to library construction we plan to separate the suspended cells from the cells that are attached to particulate material. Furthermore, we plan to separate the cells based on their size in order to get information on all members of the community, including commonly overlooked ultramicrobacteria and bacteriophages. We expect the analysis of these community subsets not only to provide an estimate of species richness and the overall genetic potential of the community, but also to identify taxa that are either particularly abundant or ecologically relevant for studied ecosystem. We further plan to learn more about the ecology of these taxa through (i) analysis of its genome segments captured as inserts of fosmid clone libraries or assembled from the short read metagenomes, (ii) through their targeted cultivation based on metagenomic data and (iii) through the analysis of single amplified genomes of these cells as recently made possible by single cell genomics. 

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