With an alarming increase in chronic diseases like childhood asthma and allergies, there is an increased focus on the exposure of young children to indoor biological and chemical air pollutants. Our study of 125 daycares throughout Norway demonstrates that the indoor mycobiome not only reflects cooccurring outdoor fungi but also includes a high abundance of yeast and mold fungi with an affinity for indoor environments.

Sampling

A list of Norwegian daycare centers was retrieved from the Norwegian Directorate of Health (Helsedirektoratet). The list was sorted alphabetically after counties and municipalities, and the first five municipalities in each county were selected for the study. Likewise, the first 3–4 daycare centers in each of these municipalities were chosen as candidate sites for dust sampling. Sampling kits containing five FLOQSwabs (Copan Italia spa, Brescia, Italy) and a questionnaire were sent to the selected daycare centers with specifications to perform dust sampling on doorframes: (1) outdoor, (2) main room and (3) bathroom. If the daycare had two different departments, we requested to repeat the sampling in (4) the main room and (5) the bathroom of the second department as well. Overall, 572 samples were retrieved from a total of 125 studied daycare centers, and upon arrival the swabs were stored at -80 ºC until DNA extraction.

DNA extraction and metabarcoding

Samples were prepared and DNA was extracted using the E.Z.N.A Soil DNA kit (Omega Biotek, Norcross, GA, USA). The tips of the swabs were placed in disruptor tubes by using a sterilized scissor. The empty swab tubes were filled with 800 µL SLX-Mlus Buffer to collect the remaining dust before being transferred to the disruptor tubes. The samples were homogenized for 2 × 1 min at 30 Hz using a TissueLyser (Qiagen, Hilden, Germany) and stored at -20 °C until further processing.

DNA extraction and metabarcoding library preparation were performed according to Estensmo et al. (10). Briefly, samples were thawed at 70 °C, followed by an incubation of 10 minutes at the same temperature and homogenized for 2 × 1 min at 30 Hz using the TissueLyser. The samples were then cooled on ice before adding 600 µL chloroform, vortexed and centrifuged at 13 000 rpm for 5 min at RT. The aqueous phase was transferred to a new 1.5 mL tube and an equal volume of XP1 Buffer was added before vortexing. The extract was transferred to the HiBind DNA Mini Column and further processed following the manufacturer's guidelines. The DNA was eluted in 50 µL Elution Buffer.

We targeted the ITS2 region with the forward primer ITS4: 5′-xCTCCGCTTATTGATATG (41) and the modified reverse primer gITS7: 5′-xGTGARTCATCGARTCTTTG (42), barcodes x ranging from 6-9 base pairs. The amplification mix contained 2 µl DNA template, 14.6 µl Milli-Q water, 2.5 µl 10x Gold buffer, 0.2 µl dNTP’s (25 nM), 1.5 µl reverse and forward primers (10 µM), 2.5 µl MgCl2 (50 mM), 1.0 µl BSA (20 mg/ml) and 0.2 µl AmpliTaq Gold polymerase (5 U/µl). DNA was amplified by initial denaturation at 95 °C for 5 min, followed by 32 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1 min. A final elongation step was included at 72 °C for 10 min. Amplicons were normalized using the SequalPrep Normalization Plate Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and eluted in 20 μL Elution Buffer. The resulting PCR products were processed into seven libraries of 96 samples using a combination of 96 tagged primers. Each library included 10 technical replicates (DNA extracts from the same 10 dust samples being amplified and sequenced independently for each library), one mock community (artificial fungal community composed of DNA in 1 ng/µL equimolar concentration from Mycena belliarum, Pycnoporellus fulgens, Serpula similis and Pseudoinonotus dryadeus), negative DNA controls (using a clean swab as starting material) and negative PCR controls. The 96 PCR products within each library were pooled, concentrated and purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, CA, USA). The quality of the purified pools was measured using Qubit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The seven libraries were barcoded with Illumina adapters, spiked with PhiX and sequenced in three Illumina MiSeq (Illumina, San Diego, CA, USA) lanes with 2 x 250 bp paired-end reads at Fasteris SA (Plan-les-Ouates, Switzerland).