Nanopore sequencing of DNA barcodes succeeds in unveilling the diversity of fungal mock communities

Abstract

Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While metabarcoding approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified nuclear ribosomal internal transcribed spacer (ITS) of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.