**2. Materials and methods**

relatively cheap and fast way of studying the diversity of microbial species by discovering representatives of novel divisions or even phyla [2] and analyzing the variation within the species by sequencing closely related genomes from the ecological microbial populations or

Historically, prokaryotic organisms were organized by classical taxonomic ranking system (species, genus, family, order, and phylum). Delineation of prokaryotic species was originally based on phenotypic information, pathogenicity, and environmental observations. Due to the high mutation level, fast replication rate, and efficient DNA exchange mechanisms, microbial organisms can easily adapt to their habitats. Genomic studies have shown that different species living in similar ecological environments show similarity at the genomic level (e.g., congruent evolution of water-living bacteria from various taxonomic origins [3]) while same pathogenic species (or symbionts) rapidly adapting to the new hosts become quite different at genomic

Next-generation sequencing technologies provide new insights into the life of microbes and their interactions with the host, but they do not classify the organisms in a traditional way.

The genomes of uncharacterized isolates of the Candidatus Arthromitus, host-specific

The number of uncharacterized species is rapidly growing in public genome collections. As of November 2014, almost half of bacterial and archaeal species in NCBI Refseq data set remain uncharacterized. (Bacteria: 3,559 uncharacterized, 7,597 total; Archaea: 162 uncharacterized,

The need for different approaches to the identification of microbial species that can take into account the advantages of the growing massive volume of genomic sequence data is being

Scientists from different disciplines (taxonomists, ecologists, and evolutionary biologists) have different interpretations of species defined by the framework of their needs and the tools they use for identification. A recent review [7] describes the history and present state of various methods of description of prokaryotic species. The authors suggest the concept of species as "a category that circumscribes monophyletic, and genomically and phenotypically coherent populations of individuals that can be clearly discriminated from other such entities by means

Comparative analysis requires a target: a coherent group of isolates with some degree of similarity defined by the goal of the study (the analysis of pathogen outbreak performed at the species level or below, while biodiversity studies use broader group such as families or phyla). Several groups have attempted to delineate the taxonomy of Archaea and Bacteria using the methods based on single-copy universally conserved markers [8-13]. Other methods

clinical studies of pathogenic bacteria.

230 Next Generation Sequencing - Advances, Applications and Challenges

level (e.g., *Buchnera aphidicola* [4], *Serratia symbiotica* [5]).

http://www.ncbi.nlm.nih.gov/genome/13597

actively discussed in the research community.

of standardized parameters."

are discussed in recent reviews [14].

399 total.)

Many novel species are described as "candidatus" or "<genus> sp."

intestinal symbionts, comprise a distinct clade within the Clostridiaceae [6].

The genomes are organized in hierarchical groups calculated with different methods. The universal ribosomal markers approach is used to build a distance tree and to define species and superspecies-level clades (genome groups). The species-level clades are further refined by using whole-genome alignments and creating tight (clonal) genome groups.
