Environmental Microbiology Reports (2016) 8(5), 774–779 doi:10.1111/1758-2229.12438 Fungal identification biases in microbiome projects Leho Tedersoo1* and Bjorn€ Lindahl2 (HTS) technologies (Fig. 1). The majority of mycology 1Natural History Museum, University of Tartu, 14a and microbiology laboratories focus on the ITS2 subre- Ravila, Tartu 50411, Estonia. gion, but the microbiome projects utilize and endorse the 2Department of Soil and Environment, Swedish primers ITS1F (Gardes and Bruns, 1993) and ITS2 University of Agricultural Sciences, Box 7014, Uppsala, (White et al., 1990), which target the ITS1 subregion SE 75007, Sweden (Prober et al., 2015; Walters et al., 2015; Metcalf et al., 2016). As judged from thousands of citations, these decades-old primers have outstandingly served the sci- SUMMARY entific community in the DNA fingerprinting and Sanger Fungi are the key players in ecosystems as well as in sequencing era. However, these primers have one or plant and human health. High-throughput molecular more mismatches to several broad fungal groups (Belle- identification of fungi has greatly progressed our main et al., 2010). For another set of ITS primers, a sin- understanding about the diversity of mutualists, sap- gle central mismatch reduced the relative abundance of rotrophs, and pathogens. We argue that the methods amplicons >10-fold in artificial communities (Ihrmark promoted by the microbiome consortia are subopti- et al., 2012). mal for detection of the most important fungal patho- Here, we highlight the critical mismatches of ITS1F gens and ecologically important degraders. We and ITS2 primers to ecologically and clinically important recommend several sets of optimized primers for fungal taxa and provide explicit evidence for their dis- analysis of fungi or all eukaryote groups based on crimination in soil fungal communities. Our ultimate pur- either short or long amplicons that cover the ITS pose is to provide alternative options for improving region as well as part of 18S and 28S rDNA. fungal HTS-based molecular identification to be globally comparable and applicable in all disciplines. Introduction Microbes play a key role in nutrient cycling and disease, Results and discussion yet the diversity and community composition of prokar- In silico tests (see Experimental procedures section) yotes, protists, and fungi are poorly known compared revealed that both the ITS1F and ITS2 primers with plants and animals. Large-scale initiatives such as completely miss Microsporidia, which are common the Human Microbiome Project (Turnbaugh et al., 2007), pathogens of a wide variety of organisms. Similarly, both the Earth Microbiome Project (Gilbert et al., 2010), and primers have multiple mismatches to Tulasnellaceae that the Microbial Earth Project [http://genome.jgi–psf.org/ constitutes the most common group of orchid mycorrhi- programs/bacteria–archaea/MEP/index.jsf] serve to pro- zal symbionts world-wide (Dearnaley et al., 2012). The vide deeper insights into the diversity and functions of same problem is inherent to all forward primers microorganisms. These consortia have established stan- within 5.8S rDNA, indicating that both ITS1 and ITS2 dardized laboratory protocols for marker-based identifi- regions separately are unsuited for identification of these cation and metagenomics. While the standards for taxa (Oja et al., 2015). The ITS2 primer possesses 30 prokaryote identification have been widely accepted, terminal mismatches to nearly all species in five early those for eukaryotes are subject to debate (Lindahl diverging fungal (sub)phyla (Fig. 2). Of these, Neocalli- et al., 2013; Geisen et al., 2015). Although the whole mastigomycotina are gut symbionts of artiodactyls, Zoo- Internal Transcribed Spacer (ITS) region has been nomi- pagomycotina and Entomophthoromycotina are obligate nated a formal barcode for fungi (Schoch et al., 2012; animal parasites, while Rozellomycota (syn. Cryptomy- Bates et al., 2013), there is no consensus about the cota) and Chytridiomycota represent the most diverse selection of ITS subregions (ITS1 or ITS2) and primers and abundant groups of aquatic fungi (Richards et al., for identification based on high-throughput sequencing 2012; Hassett and Gradinger, 2016). Besides chytrids, members of the Venturiales (Ascomycota), Exobasi- Received 5 May, 2016; accepted 17 July, 2016. *For correspon- dence. E-mail [email protected]; Tel. (1372) 56654986; Fax diales, Ustilaginales and Entylomatales (all Basidiomy- (1372) 7376222.. cota) are important plant pathogens that are all missed VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd Fungal identification biases in microbiome projects 775 Fig. 1. Primer map of the ITS region indicating the discussed primers. Primers in red denote those used by the microbiome consortia; [I] indi- cates a common intron site. by the ITS2 primers. Most species within the Omphalo- microflora and/or pathogens (Hoffmann et al., 2013; taceae family and the related genus Mycena, which are Hallen-Adams et al., 2015). among the globally most diverse and abundant saprotro- To test the differential performance of primers in the phic decomposers of plant litter (Tedersoo et al., 2014; mismatching taxa, we re-analyzed a soil fungal data Sterkenburg et al., 2015), have a terminal mismatch at from natural habitats of Papua New Guinea that was the 30 end of the ITS2 primer. In many environments, generated by Illumina MiSeq sequencing (Tedersoo exclusion of these groups by the ITS2 primer may et al., 2015a). We selected this data set, because it was potentially lead to wrong conclusions about the distribu- the only one generated with >1 primer combination for tion of fungal functional guilds and their contribution to both ITS1 and ITS2 regions. Taxonomic analysis of the ecosystem processes, such as carbon turnover during soil HTS data revealed that nearly half of the groups litter decomposition. Terminal and near-terminal ITS2 with terminal mismatches (Ustilaginales, Entylomatales, primer mismatches are also alarming for human gut and Malasseziales, Exobasidiales, and Ascosphaeraceae) skin microbiota, including opportunistic pathogens, such are completely missing in the ITS1 data subset, while as true yeasts Galactomyces geotrichum and Yarrowia others are reduced 15-fold on average (Fig. 3). Except lipolytica and basidio-yeasts belonging to Malasseziales for Rozellomycota, taxa with near-terminal mismatches and Tremellales s.lat. (LaTuga et al., 2011; Huffnagle are less affected (three-fold on average). Near-terminal and Noverr, 2013; Hallen-Adams et al., 2015). Among and central mismatches have even stronger effect on the latter group, Cryptococcus neoformans causes lethal the performance of the ITS1F primer. For example, rela- cryptococcosis in immunosuppressed patients. Emmon- tive abundance of Saccharomycetaceae, Mucoromyco- sia parva and Ajellomyces capsulatus (Ajellomyceta- tina (excl. Umbelopsidaceae) and Umbelopsidaceae is ceae) are causal agents of adiaspiromycosis and reduced by a factor of 9.0, 117.7, and 16.8, respectively. histoplasmosis, respectively. The ITS1F primer has mul- Taken together, our analyses indicate that the diversity tiple central mismatches to nearly all taxa in the subphy- of many ecologically and functionally important fungal lum Mucoromycotina (Fig. 2) that includes molds and groups is severely underestimated by the primers used opportunistic human pathogens. This primer has also by microbiome consortia. Moreover, the relative near-terminal mismatches to much of the Saccharomy- performance of mismatching primers is unpredictable, cetes, including Saccharomyces cerevisiae (baker yeast) because primer annealing depends on PCR conditions, and Candida spp. – abundant members of the normal type of polymerase, the nature of mismatching Fig. 2. Template mismatches of the ITS1F and ITS2 primers. Only groups with >10% mismatching accessions are indicated. Taxa with single mismatches in the central area and 50 end are not shown for the ITS2 primer; taxa with single mismatches in the 50 end are not shown for the ITS1F primer. VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 8, 774–779 776 L. Tedersoo and B. Lindahl Entomophthoromycotina *** fungi. Fungi are highly dominant in soil (90–93% of Mycena *** eukaryote ITS sequences; Tedersoo et al., 2015a; 2016a), Omphalotaceae * Ustilaginales *** wild boar feces (89%; L. Tedersoo, unpublished), leaves Entylomatales *** (39%; Remmelgas, 2016), and non-ectomycorrhizal roots Malasseziales *** (7%; Oja et al., 2015), based on nearly universal eukary- Exobasidiales *** Ajellomycetaceae ote primers. Therefore, fungal diversity may be captured Ascosphaeraceae *** using broader, universal primers, which also enable rough Tremellales *** Leucosporidiales *** estimates of relative abundance across broad eukaryote Atractiellomycetes *** groups (Tedersoo et al., 2016a). Furthermore, additional Chytridiomycota *** peptide-nucleic acid PCR clamps can be used to reduce Rozellomycota *** Venturiales *** amplification of other organisms such as the host species Saccharomycetes *** (Lundberg et al., 2013), but this approach is seldom Serendipitaceae ** Gomphaceae applied in environmental microbiology. (-)2 1 2 4 8 16 32 64 128 256 512 For the above reasons, we advocate the use of the ITS2 subregion and a degenerate version of the ITS4 Ratio of ITS2/ITS1 sequence proportions primer (White et al., 1990) in combination with modified Fig. 3. Relative sequence