Each day more genome data are available and so are the number of bioinformatic resources, thus researchers are faced with making the right choice of analytical tools. In addition, with decreasing prices for genome sequencing, comparative genomics is a continuously growing field, especially for fungal genomes given their small sizes (i.e. average genome size 1C = 46 Mb). Given these opportunities and aiming to gain novel insights into the evolution of mutualisms, we focused on the cultivars of fungus-growing ants (Hymenoptera: Formicidae: Attini) and a free-living relative, and found that current methodology needs refining. By using different genome assemblers, we show that the genome assembly size depends on what software is used. This in turn impacts gene number predictions, with higher gene numbers correlating positively with genome assembly size. Currently, the majority of fungal genome sizes available are based on estimations derived from whole genome assemblies rather than from the more accurate technique of flow cytometry. Here, we employed flow cytometry to accurately estimate the haploid genome sizes of three ant fungal symbionts using the fungus Pleurotus ostreatus (Jacq.) P. Kumm. (1871) as a calibration standard. We found that published genome sizes based on earlier genome assemblies are 2.5 to 3-fold larger than our estimates based on flow cytometry. We therefore recommend that these measurements are used to pre-calibrate genome assemblies, to avoid incorrect estimates of genome sizes.
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