DNA metabarcoding has become a powerful approach for analysing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers – like the fungal ITS region, may include considerable intraspecific variation, which can lead to oversplitting of species during DNA metabarcoding analyses. Here we assessed the effects of intraspecific sequence variation in DNA metabarcoding by analysing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS) coupled with error correction with the software dada2. All the eleven species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, typically occurring in low frequencies, were probably due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS‐based fungal community analyses, but an extra clustering step is needed to approach species‐level resolution.

Background Children spend considerable time in daycare centers in parts of the world and are exposed to the indoor micro- and mycobiomes of these facilities. The level of exposure to microorganisms varies within and between buildings, depending on occupancy, climate, and season. In order to evaluate indoor air quality, and the effect of usage and seasonality, we investigated the spatiotemporal variation in the indoor mycobiomes of two daycare centers. We collected dust samples from different rooms throughout a year and analyzed their mycobiomes using DNA metabarcoding. Results The fungal community composition in rooms with limited occupancy (auxiliary rooms) was similar to the outdoor samples, and clearly different from the rooms with higher occupancy (main rooms). The main rooms had higher abundance of Ascomycota, while the auxiliary rooms contained comparably more Basidiomycota. We observed a strong seasonal pattern in the mycobiome composition, mainly structured by the outdoor climate. Most markedly, basidiomycetes of the orders Agaricales and Polyporales, mainly reflecting typical outdoor fungi, were more abundant during summer and fall. In contrast, ascomycetes of the orders Saccharomycetales and Capnodiales were dominant during winter and spring. Conclusions Our findings provide clear evidences that the indoor mycobiomes in daycare centers are structured by occupancy as well as outdoor seasonality. We conclude that the temporal variability should be accounted for in indoor mycobiome studies and in the evaluation of indoor air quality of buildings.

BackgroundThe preterm infant gut microbiota is vulnerable to different biotic and abiotic factors. Although the development of this microbiota has been extensively studied, the mobilome-i.e. the mobile genetic elements (MGEs) in the gut microbiota-has not been considered. Therefore, the aim of this study was to investigate the association of the mobilome with birth weight and hospital location in the preterm infant gut microbiota.MethodsThe data set consists of fecal samples from 62 preterm infants with and without necrotizing enterocolitis (NEC) from three different hospitals. We analyzed the gut microbiome by using 16S rRNA amplicon sequencing, shot-gun metagenome sequencing, and quantitative PCR. Predictive models and other data analyses were performed using MATLAB and QIIME.ResultSThe microbiota composition was significantly different between NEC-positive and NEC-negative infants and significantly different between hospitals. An operational taxanomic unit (OTU) showed strong positive and negative correlation with NEC and birth weight, respectively, whereas none showed significance for mode of delivery. Metagenome analyses revealed high levels of conjugative plasmids with MGEs and virulence genes. Results from quantitative PCR showed that the plasmid signature genes were significantly different between hospitals and in NEC-positive infants.ConclusionOur results point toward an association of the mobilome with hospital location in preterm infants.

Background: Children spend considerable time in daycare centers in parts of the world, and are exposed to the indoor micro- and mycobiomes of these facilities. The level of exposure to microorganisms varies within and between buildings, depending on occupancy, climate and season. In order to evaluate indoor air quality, and the effect of usage and seasonality, we investigate the spatiotemporal variation in the indoor mycobiomes of two daycare centers. We collected dust samples from different rooms throughout a year and analyzed their mycobiomes using DNA metabarcoding. Results: The fungal community composition in rooms with limited occupancy (auxiliary rooms) was similar to the outdoor samples, and clearly different from the rooms with higher occupancy (main rooms). The main rooms had higher abundance of Ascomycota, while the auxiliary rooms contained comparably more Basidiomycota. We observed a strong seasonal pattern in the mycobiome composition, mainly structured by the outdoor climate. Most markedly, basidiomycetes of the orders Agaricales and Polyporales, mainly reflecting typical outdoor fungi, were more abundant during summer and fall. In contrast, ascomycetes of the orders Saccharomycetales and Capnodiales were dominant during winter and spring. Conclusions: Our findings provide clear evidences that the indoor mycobiome in daycare centers are structured by occupancy as well as outdoor seasonality. We conclude that the temporal variability should be accounted for in indoor mycobiome studies and in the evaluation of indoor air quality of buildings.

DNA metabarcoding has become a powerful approach for analyzing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers -like the fungal ITS region, may include considerable intraspecific variation, which can lead to over-splitting of species during DNA metabarcoding analyses. Here we assess the effects of intraspecific sequence variation in DNA metabarcoding, by analyzing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS), coupled with error correction with the software DADA2. All focal species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, often occurring in low frequencies, were likely due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS-based fungal community analyses, but an extra clustering step is needed to approach species-level resolution.