ability to now obtain large amounts of molecular data What are the

ability to now obtain large amounts of molecular data. What are the relative strengths and weaknesses of the various types of data that could be used? What specific data do you think should be used for defining bacterial species?

There are numerous experimental methods by which a researcher can define bacterial species, broadening and "fixing" phylogenetic trees in bacterial species: some new and some old, some expensive and some cheap, some more effective than others. It has been a struggle to differentiate accurately among bacterial species as there are so many extenuating circumstances that could cause error in data.

Below, I will discuss the different strengths and weaknesses in which phenotypic characterizations, hybridization experimentations, and new methods using molecular and genomic data aid in delineating bacterial species.

In an experiment, researchers examined, using Acinetobacter as a model, whether phenotypic approaches and DNA-DNA hybridization could be fully replaced by genome sequence analyses (Chan et. al 2012). Vandamme and Peeters referred to MLSA as one of the methods that could be applied as a tool to replace DNA-DNA hybridization in species delineation as well (Vandamme et al.

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2014).

Using core genome phylogenetic analysis and average nucleotide identity via BLASTn bacterial species can be defined and classified appropriately. Bacterial species are, by definition, monophyletic groups with genomes that possess 95% pair-wise average nucleotide identity or more. These methods provide "a scalable and uniform approach that works for both culturable and non-culturable species," a significant strength of this genomic and molecular analysis; in addition, it is also much faster and less expensive than traditional taxonomic methods that have been used previously.

These genomic methods are also easily replicable and transferable among research institutions. Finally, these methods fall in line with Darwin's vision of classification, another strength of these molecular processes that some are convinced will replace DNA-DNA hybridization and phenotypic calculations (Chan et. al 2012).

As stated, the "gold standard" is 70% DNA-DNA hybridization. In simplistic terms, hybridization occurs via temperature increase of a specific DNA fragment after PCR amplification causing the separation of the DNA into single strands. By combining two sample bacterial DNA, the new DNA, tagged, will undergo 70% hybridization if the same species. This "gold standard" of bacterial species determination and definition is the overall genome similarity determined by DNA-DNA hybridization, a technically rigorous yet sometimes variable method that may produce inconsistent results, a major weakness of the much used method. (Colston et al. 2014 & Rossello-Mora et al. 2015).

One significant strength of genomic sequencing and molecular data includes that the data and computing programs can be readily shared among scientists, aiding in standardizing methodologies and phylogenetic information in the new age of biological taxonomic information as these databases become more and more readily available for further bacterial classification. This standardization can resolve "conflicting identifications by providing greater uniformity in an overall analysis" (Colston et al. 2014). Additionally, researchers made new discoveries about the overall relationships of the Aeromonas species with the phylogeny generated from the expanded core and the housekeeping genes, demonstrating that genetic analysis can be much more accurate in phylogeny building in comparison to DNA-DNA hybridization and phenotypic characteristics that were used to build them previously (Colston et al. 2014).

Similarly, a significant strength of DNA-DNA hybridization is that DNA content of a cell is invariant regardless of growth conditions; therefore, it is more objective and is invariable from laboratory to laboratory, experiment to experiment. However, if nucleotide sequence similarity is less than 15%, binding and hybridization will not occur. There is no resolution above the genus level. DNA-DNA hybridization is quite labor intensive and very expensive to undergo due to the special equipment and complex reagents. While cost does not necessarily determine how effective this method is in determining species differentiation in bacterial communities, it is still very important to take the price into account as budget is a large consideration in receiving grants for further research.

However, with this immense strength of genetic and molecular data in delineating bacterial species, expanded core phylogenies have been found to, "using the genes that are present in over 90% of the 56 known strains of Aeromonas," hold inherent biases which could give rise to inaccurate results whilst depicting organismal phylogenies. Because gene transfer between divergent groups has the potential to create phylogenetic trees from linked data sets that do not reflect the vertical inheritance of said bacterial DNA fragments, errors in any bacterial data collection can be tainted, especially found in this example of the use of expanded core phylogenetic analysis (Colston et al. 2014 & Lapierre et al. 2012).

The main limitation to a universal measure for all prokaryotic taxa is the lack of genes that are widely distributed in all taxa; recent estimates suggest that there are