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 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 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 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 (), 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 (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 *** 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 *** (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 abundance ratio of averaged gITS7 (Ihrmark et al., 2012) or ITS3ngs mixes (Tedersoo ITS3ngsmix-ITS4ngs and gITS7-ITS4ngs amplicons to averaged et al., 2014; Table 1) when applying current second- ITS1Fngs-ITS2 and ITS1ngs-ITS2 amplicons (n 5 26). Relative per- formance of the ITS2 primer was compared by contrasting the aver- generation sequencing technologies that produce short age results of amplicons ITS1ngs-ITS2 and ITS1Fngs-ITS2 against reads. The proposed primer ITS4ngsUni accounts for the average of ITS3mix-ITS4ngs and gITS7-ITS4ngs. The contrasts were statistically analyzed by testing the differences in relative nearly all mismatches in fungi and most eukaryotes, abundance of sequences belonging to targeted taxa across 26 soil except Foraminifera and Microsporidia. Because HTS samples using a series of two-way ANOVAs, treating samples as technologies evolve rapidly in terms of read length, quali- blocking factors. Error bars represent standard error. Zoopagomy- cotina, Neocallimastigomycotina, Neolectomycetes, Morchellaceae, ty, and throughput, there is a great potential to cover the Yarrowia, and Galactomyces were present in <5% of samples and entire ITS region for more accurate species-level identifi- were not tested. Asterisks indicate significant differences between cation (Schoch et al., 2012; Koljalg~ et al., 2013). The ITS1 and ITS2 region, accounting for false discovery rate using Benjamini–Hochberg correction (*P < 0.05; **P < 0.01; combination of modified ITS1 and ITS1F forward primers ***P < 0.001). Negative side of 2-base logarithmic scale indicates (Oja et al., 2015) and the ITS4ngsUni reverse primer ITS1/ITS2 ratio. serve as strong candidates for HTS analysis of the full ITS barcode (600–700 bases) in fungi. For longer ampli- and neighboring nucleotides, sample complexity, etc. cons, the ITS4ngsUni could be substituted by optimized (Kanagawa, 2003). primer TW13 (1200–1300 bases) or TW14 (1500–1600 Previous studies indicate that the ITS1 region yields bases; White et al., 1990) that are highly conserved fewer OTUs and lower phylogenetic richness of fungi across eukaryotes. While these 28S rDNA primers allow compared with the ITS2 region (Mello et al., 2011; Op de better taxonomic assignments for and former Beeck et al., 2014; Tedersoo et al., 2015a,b). While the zygomycetes, a portion of 18S would enable to improve overall variability of ITS1 and ITS2 regions is comparable identification of the early diverging zoosporic fungal line- across fungi (Nilsson et al., 2008), PCR and sequencing ages. For this option, an optimized version of a universal biases may recover lower fungal diversity using the ITS1 eukaryote forward primer ITS9MUN (Egger, 1995) could marker. Besides primer bias, multiple species from sever- be used in combination with ITS4ngsUni (750–850 al ascomycete classes possess an intron downstream of bases) or primers further downstream in 28S rDNA. How- the ITS1F priming site (Bhattacharya et al., 2000). In ever, due to greater PCR efficiency, short amplicons are addition, the ITS1 region has greater length heterogeneity less prone to amplification biases, and probably yield among eukaryote phyla and fungal taxa (Tedersoo et al., more accurate quantitative insights (Ihrmark et al., 2012). 2015a; Wang et al., 2015). Among ecologically and eco- We advocate that metagenomic approaches are unlikely nomically important fungi, Cantharellus and Boletaceae to replace amplicon-based surveys of eukaryotes in the species exhibit excessively long ITS1 regions, but this is near future, because of their low biomass and problems also true for the ITS2 region of some Boletaceae. Both with sequence assignment to OTUs and reference taxa the intron and oversized ITS1 may render these taxa out- (Tedersoo et al., 2015a,b; 2016b). competed in amplification and emPCR steps that discrim- inate against longer templates. Experimental procedures The capacity of HTS technologies to cover species diversity to saturation (Caporaso et al., 2012) calls into We used the aligned 18S, 5.8S, and 26S rDNA sequence question the necessity of -specific primers for data set (Bengtsson-Palme et al., 2013) for initial

VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 8, 774–779 Fungal identification biases in microbiome projects 777

Table 1. Recommended primers for high-throughput identification of fungi.

Melting Primer Sequence (50-30) temperature (8C) Suitable linkera Target

ITS1ngs TCCGTAGGTGAACCTGC 54 AA Many eukaryotes (Fungi: except ) ITS1 (original) TCCGTAGGTGAACCTGCGG 62 Many eukaryotes (Fungi: except Sordariomycetes, Microsporidia ITS1Fngs GGTCATTTAGAGGAAGTAA 52 GA Fungi (many exceptions) ITS1F (original) CTTGGTCATTTAGAGGAAGTAA 60 Fungi (many exceptions) ITS3ngsmix1-5 CANCGATGAAGAACGYRGa 55 AT Fungi, plants ITS3 (original) GCATCGATGAAGAACGCAGC 62 Fungi (many exceptions), plants gITS7ngs GTGARTCATCRARTYTTTG 52 GG Fungi, plants, many protists gITS7 (original) GTGARTCATCGARTCTTTG 54 Fungi (many exceptions), plants ITS4ngsUni CCTSCSCTTANTDATATGC 55 CG Eukaryotes (except Foraminifera, Microsporidia) ITS4 (original) TCCTCCGCTTATTGATATGC 58 Eukaryotes (many exceptions) ITS9MUNngs TACACACCGCCCGTCG 54 GA Eukaryotes ITS9MUN (original) TGTACACACCGCCCGTCG 60 Eukaryotes TW13 GGTCCGTGTTTCAAGACG 56 AG Eukaryotes (except dictyostelids) TW14 (original) GCTATCCTGAGGGAAACTTC 60 Eukaryotes (many exceptions) TW14ngs CTATCCTGRGRGAAAYTTC 55 GC Eukaryotes

Note: The primers with “ngs” suffix were designed recently (Tedersoo et al., 2014; Oja et al., 2015) by shortening the corresponding original primers to achieve similar melting temperature and reduce the proportion of mismatching nucleotides in critical positions (underlined bases; see Tedersoo et al., 2016a for details). a. An inert sequence between identifier tag and primer for securing equal performance of tag and primer combination and avoiding secondary structure. b. consensus of CATCGATGAAGAACGCAG, CAACGATGAAGAACGCAG, CACCGATGAAGAACGCAG, CATCGATGAAGAACGTAG, CATCGA TGAAGAACGTGG. monitoring of primer-template mismatches across the fun- primer mismatches in previous studies and blurred dif- gal kingdom and identified specific taxonomic groups ferences among primers, especially those in 30 18S and (genus to phylum level) where mismatches were common 50 28S rDNA. By including problematic reference (>5% of accessions) and not attributable to incomplete or sequences, 5–30% of entries were mismatched that fall erroneous data. Compromised quality was assigned to into the range reported in most previous studies. sequences that had the primer sites only partly covered Removal of inappropriate sequences reduced the mis- and that were obviously amplified and sequenced using match frequency five-fold to 10-fold and provided clear the primers we evaluated (as seen from matching start and consistent taxonomic patterns for primer bias. In and end of query and reference sequences). Ambiguous reporting mismatches, we define positions in the primers nucleotides, unique single nucleotide indels and unique as terminal (two last 30 nucleotides), near-terminal (third substitutions (occurring once or <0.1% for a taxonomic and fourth nucleotide from 30 end), central (fifth in 30 end group) in conserved regions were also considered indica- to fifth in 50 end), and 50 (four nucleotides in 50 end). tive of low sequence quality. These sequences were Based on the above rationale of sequence data quali- excluded from primer-template mismatch evaluation. ty filtering, we removed compromised sequences in data To determine additional mismatching taxa, we per- sets of all eukaryote kingdoms (Bengtsson-Palme et al., formed blastN searches (parameters: word size 5 7; 2013) for designing improved primers. Based on the par- match score 5 1; mismatch score 523; gap opening tial alignments of the 18S, ITS, and 26S rDNA, we cost 5 5; gap extension cost 5 2) using all single- designed modifications to the existing primers to cover mismatched variants of the primer sequence against all either all fungi or all eukaryotes (still, with certain fungi in the entire International Nucleotide Sequence exceptions), depending on the level of primer site Databases collaboration (INSDc) (accessed 2016-01- conservation. 20). The relative frequency of mismatching entries in these problematic taxa was determined by calculating the proportion of mismatching primer variants among all Acknowledgements hits using taxonomically filtered blastN searches against We thank R.H. Nilsson for providing the 18S-ITS-28S data these particular taxonomic groups. We used the above sets of eukaryotes and discussion about ITS sequence described criteria to disregard references of low quality. length; T.D. Bruns, J.W. Taylor, and J. Gilbert for construc- Inclusion of low-quality, non-fungal and mitochondrial tive comments on an earlier version of the manuscript. L.T. reference sequences in in silico tests of primer suitability acknowledges funding from the Estonian Science Founda- for fungi seem to have severely overestimated the true tion grants PUT171, RMK, KIK, and EcolChange.

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