Ion torrent PGM as tool for fungal community analysis : a case study of endophytes in eucalyptus grandis reveals high taxonomic diversity

摘要

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendousdiversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungalcommunities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy planttissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent offungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used infungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomalRNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the IonTorrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation ofthe results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering andfiltering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had amarginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on theassignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly byfamilies in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductorsequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potentialpitfalls in subsequent data analyses when using a technology with relatively short read lengths.