Comprehensive Fungal Community Analysis of House Dust Using Next-Generation Sequencing

Abstract

Fungal community analyses in homes have been attracting attention because fungi are now generally considered to be allergens. Currently, these analyses are generally conducted using the culture method, although fungal communities in households often contain species that are difficult to culture. In contrast, next-generation sequencing (NGS) represents a comprehensive, labor- and time-saving approach that can facilitate species identification. However, the reliability of the NGS method has not been compared to that of the culture method. In this study, in an attempt to demonstrate the reliability of this application, we used the NGS method to target the internal transcribed spacer 1 (ITS1) in the fungal genome, conducted fungal community analyses for 18 house-dust samples and analyzed fungal community structures. The NGS method positively correlated with the culture method regarding the relative abundance of Aspergillus, Penicillium, Cladosporium and yeasts, which represent the major fungal components found in houses. Furthermore, several genera, such as Malassezia, could be sensitively detected. Our results imply that the reliability of the NGS method is comparable to that of the culture method and indicates that easily available databases may require modifications, including the removal of registrations that have not been sufficiently classified at the genus level.

Keywords: ITS region; fungal community analysis; house dust; next-generation sequencing.

Figure 1

Rarefaction curves of all samples. (a) Rarefaction curves for SEH3 to SEH34 and (b) rarefaction curves for SEH37 to SEH61. Dotted lines are the duplicates of solid line samples in various colors.

Figure 2

Dendrogram of all samples according to the unweighted pair group method with arithmetic means (UPGMA) based on the beta diversity of community structures.

Figure 3

Taxonomic compositions of the fungal communities in 36 PCR duplicates of house dust samples. (a) Without the removal of database sequences classified at the genus level; (b) with those sequences removed. Gray bar indicates the ‘Others’ category defined as the occupancy rate of the total remaining species other than the 19 genera shown in this figure. Red bar indicates the ‘unidentified at the genus-level category’ defined as the assignment to database registrations that are not classified at the genus level. Purple bar indicates the ‘unassigned’ category defined as the failure to be assigned to any database sequences with more than a 98% homology.

Figure 4

Comparisons of fungal occupancy rates between the next-generation sequencing (NGS) and culture methods. The bars are arranged from left to right by increasing total fungal counts/1 g house dust.