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An Amplicon-Based Sequencing Approach for the Study Of Journal of Xenobiotics 2018; volume 8:7810 An amplicon-based sequencing son of the performance of ITS1 and ITS2 in approach for the study of determining the fungal diversity of Correspondence: Hamza Mbareche, Centre de bioaerosols. Recherche de l’Institut Universitaire de aeromycology Cardiologie et de Pneumologie de Québec, Quebec City (QC), Canada. E-mail: [email protected] Hamza Mbareche,1,2 Marc Veillette,1 Materials and Methods 3 Guillaume J. Bilodeau, Various air samples were collected: Key words: Amplicon-based sequencing 1,2 Caroline Duchaine composting sites, biomethanization facili- approach; Aeromycology. 1Centre de Recherche de l’Institut ties and dairy farms. In each environment, Contributions: H.M. designed the study, per- the sampling sites were chosen according to Universitaire de Cardiologie et de formed all experiments, including field sam- Pneumologie de Québec, Quebec City where workers were most exposed to pling, analyzed the data and wrote the paper; (QC); 2Département de Biochimie, de bioaerosols. Each composting plant treats M.V. performed field sampling and designed Microbiologie et de Bio-informatique, different raw materials: household green the study; G.J.B. and C.D. designed the study; waste (domestic), manure and hay (vege- and all authors edited the manuscript. Faculté des Sciences et de Génie, tal), and pig carcasses and placenta (ani- Université Laval, Quebec City (QC); Conflict of interest: the authors declare no 3 mal). All of the composting plants were Pathogen Identification Research Lab, located in the province of Quebec, Canada. potential conflict of interest. Canadian Food Inspection Agency Samples were also collected from two (CFIA), Ottawa, Canada Funding: this work was supported by the biomethanization facilities. One facility Institut de Recherche Robert-Sauvé en Santé processes primary and secondary sludge et en Sécurité du Travail (2014-0057). from wastewater treatment plants, as well as Introduction industrial waste. The second one handles Conference presentation: part of this paper municipal waste from domestic sources. Air was presented at the Saint-Lawrence Chapter- Fungal spores are ubiquitous in indoor samples were collected from five dairy SETAC Meeting, June 2018, Québec, Canada. and outdoor air and their diversity and farms in Eastern Canada during summer Receivedonly for publication: 6 September 2018. biomass quantity vary depending on the 2016. The buildings at each farm exhibited Accepted for publication: 17 October 2018. geographical location, environmental con- differences in building type and building ditions, and the presence of sources such as characteristics (age, volume, ventilation), This work is licensed under a Creative hay, agricultural crops or growth on build- the number of animals presents (cows), the 1,2 useCommons Attribution NonCommercial 4.0 ing material. Fungal exposure can be methods of milking (automatic or manual) License (CC BY-NC 4.0). explained by the presence of decaying and types of animal feed animals were materials like, peat, wood dust, manure, ©Copyright H. Mbareche et al., 2018 3 given. For the three environments, and dur- biosolids, and organic wastes like compost. ing each visit, three samples were taken: at Licensee PAGEPress, Italy Journal of Xenobiotics 2018; 8:7810 Waste treatment plants and dairy farms are the beginning, the middle and the end of 4-6 doi:10.4081/xeno.2018.7810 locations with high fungal exposure. The work shifts. health effects linked to this exposure are A liquid cyclonic impactor Coriolis µ® likely underestimated due to the presence of (Bertin Technologies, Montigny-le- 7 undocumented fungi. Culture methods are Bretonneux, France) was used for collecting ITS2 for metabarcoding analyses of fungal still widely used to describe fungal aerosols. air samples. The sampler was set at 300 aerosol populations? [unpublished materi- However, there are inherent biases associat- L/min for 10 minutes (3m3 of air per sam- als]). ed with these methods when applied to fun- ple) and placed within 1-2 meters of the The bioinformatics workflow used to gal diversity analyses because most fungal source. The air flow in the sampler creates a treat the sequences and analyze the diversi- species are difficult to isolate from culture. vortex through which air particles enter the ty was based on the one previously Amplicon-based sequencing is a good alter- Coriolis cone and are impacted in the liquid. described study.4 native that offers a more detailed analysis of Fifteen milliliters of a phosphate buffer the microbial composition of aerosolNon-commercial sam- saline (PBS) solution with a concentration ples due to the millions of sequences pro- of 50 mM and a pH of 7.4 were used to fill Results and Discussion duced. Amplicon-based sequencing the sampling cone. approaches depend on the critical choice of A MoBio PowerLyser® Powersoil® In compost samples, 3 871 313 raw which DNA region is used as the barcode. Isolation DNA kit (Carlsbad, CA, U.S.A) reads clustered into 1 208 OTUs Universal markers are based on different was used to extract the total genomic DNA (Operational Taxonomic Units) after quality criteria such as their presence across taxa from the samples following the manufactur- filtering for ITS1, and 3 680 926 raw reads and sufficient sequence variation between er’s instructions. Then, DNA was eluted in clustered into 772 OTUs for ITS2. In taxa. For fungi, the internal transcribed a 100µl MoBio buffer and the isolated DNA biomethanization samples, 675,642 raw spacer (ITS) region of rDNA is considered samples were stored at -20°C until subse- reads clustered into 1142 OTUs and the best barcode for most fungal groups.8-10 quent analyses. 730,688 sequences into 330 OTUs for ITS1 The ITS region is composed of three parts: Amplification of the fungal ITS1 and and ITS2, respectively. In dairy farm sam- ITS1 - 5.8S - ITS2. The limitations imposed ITS2 regions, equimolar pooling and ples, 354,262 sequences led to 1015 OTUs by the sequencers in term of amplicon size sequencing were performed at the Genomic and 310,362 sequences led to 218 OTUs for forces the use of ITS1 or ITS2 to study fun- analysis platform (IBIS, Université Laval, ITS1 and ITS2, respectively. gal diversity of bioaerosols. Quebec City, Canada). Detailed information The ITS1 barcode produced significant- The objective of this work is to use air about the protocols used for MiSeq Illumina ly higher richness and diversity measures samples from waste treatment facilities and Sequencing is presented in the original (Observed OTUs, Chao1, Shannon, dairy farms to make a systematic compari- study report (Mbareche et al., 2018. ITS1 or Simpson) per sample compared to the ITS2 [page 10] [Journal of Xenobiotics 2018; 8:7810] Article barcode in the three environments (data not shown). These results suggest ITS1 is a bet- ter diversity indicator than ITS2. The taxo- nomic profiles were analyzed more careful- ly to compare classes of fungi across sam- ples according to the barcode used in each one of the three environments studied. The results presented in Figure 1 include all three environments combined. Of the 20 classes of fungi represented, Dothideomycetes, Eurotiomycetes, Saccharomycetes, Sordariomycetes and Agaricomycetes are the most dominant accounting for 90% of the total relative abundance. The most striking difference between ITS1 and ITS2 was in the class Saccharomycetes. It was 2.5 times more abundant in results from ITS2 (20%) com- pared to ITS1 (8%). Of the less abundant taxa, Wallemiomycetes, Exobasidiomycetes and Taphrinomycetes were detected only when the ITS1 region was used and Glomeromycetes, Tritirachiomycetes, Mucoromycotina, Rozellomycota and Figure 1. Relative abundance of the classes of fungi detected in the three environments Lecanoromycetes were detected only by (compost, biomethanization and dairy farms). onlySamples were compared based on the bar- ITS2. Saccharomycetes were detected in all code used. The Blastn tool and the UNITE database were used for taxonomy assignment. three environments when the ITS2 region was used while no Saccharomycetes were detected with the ITS1 region. This discrep- ancy may be linked to a 3’ terminal mis- use match associated with ITS1 primers.11 For example, the ITS1F forward primer specific to fungi is known to have mismatches with some classes of fungi including Chytridiomycota, Saccharomycetes and some genera of Dothideomycetes. The occurrence of introns between primer sites in many taxonomic groups of Ascomycota could also explain the difference in results obtained using the ITS1 and ITS2 regions.12-14 Figure 2 presents a summary of the species that were consistently (in the three environments) more abundant when either ITS1 or ITS2 was used (based on Man- Whitney U test). Abundance comparisonsNon-commercial for all of the species in Figure 2 are signifi- cant at P<0.005. The fungal species that were found exclusively when used with either the ITS1 or ITS2 barcode should be examined more closely when designing a study of fungal diversity in bioaerosols. The differential abundance results presented herein are not exhaustive. All of the infor- mation is presented in the supporting mate- rial associated with the original study report (Mbareche et al., 2018. ITS1 or ITS2 for metabarcoding analyses of fungal aerosol populations? [unpublished materials]). Primer mismatch could explain the poten- Figure 2. Fungal species with statistically significant differential abundances across sam- tial biases linked to the taxa identified only ples from compost, biomethanization and dairy farms targeting ITS1 or ITS2 barcodes. 15,16 From the bottom to the top: 15 species that were consistently (throughout the three envi- by ITS1 or ITS2 but not both. ronments) more abundant using ITS1 and 10 that were consistently more abundant Other factors may impact the perfor- using ITS2. mance of ITS1 and ITS2 as fungal barcodes [Journal of Xenobiotics 2018; 8:7810] [page 11] Article in amplicon-based sequencing studies. For book: measurement, dosimetry and sal DNA barcode marker for Fungi. example, the GC content is known to have health effects.
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