Increased sequencing depth does not increase captured diversity of arbuscular mycorrhizal fungi
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The arrival of 454 sequencing represented a major breakthrough by allowing deeper sequencing of environmental samples than was possible with existing Sanger approaches. Illumina MiSeq provides a further increase in sequencing depth but shorter read length compared with 454 sequencing. We explored whether Illumina sequencing improves estimates of arbuscular mycorrhizal (AM) fungal richness in plant root samples, compared with 454 sequencing. We identified AM fungi in root samples by sequencing amplicons of the SSU rRNA gene with 454 and Illumina MiSeq paired-end sequencing. In addition, we sequenced metagenomic DNA without prior PCR amplification. Amplicon-based Illumina sequencing yielded two orders of magnitude higher sequencing depth per sample than 454 sequencing. Initial analysis with minimal quality control recorded five times higher AM fungal richness per sample with Illumina sequencing. Additional quality control of Illumina samples, including restriction of the marker region to the most variable amplicon fragment, revealed AM fungal richness values close to those produced by 454 sequencing. Furthermore, AM fungal richness estimates were not correlated with sequencing depth between 300 and 30,000 reads per sample, suggesting that the lower end of this range is sufficient for adequate description of AM fungal communities. By contrast, metagenomic Illumina sequencing yielded very few AM fungal reads and taxa and was dominated by plant DNA, suggesting that AM fungal DNA is present at prohibitively low abundance in colonised root samples. In conclusion, Illumina MiSeq sequencing yielded higher sequencing depth, but similar richness of AM fungi in root samples, compared with 454 sequencing.
KeywordsArbuscular mycorrhiza Illumina sequencing 454 Sequencing Community diversity
This research was funded by grants from the Estonian Research Council (grant IUT 20-28), the European Regional Development Fund (Centre of Excellence EcolChange) and ERA-NET Cofund BiodivERsA project SoilMan. Preparatory procedures for 454 sequencing were performed by BiotaP Ltd. (Tallinn, Estonia). RA and MR were supported by the EU ERDF through the Estonian Centre of Excellence in Genomics and Translational Medicine (project no. 2014-2020.4.01.15-0012) and by the Estonian Ministry of Education and Research (institutional grant IUT34-11). We are grateful to the Estonian Genome Centre for technical support.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Davison J, Moora M, Öpik M, Adholeya A, Ainsaar L, Bâ A, Burla S, Diedhiou AG, Hiiesalu I, Jairus T, Johnson NC, Kane A, Koorem K, Kochar M, Ndiaye C, Pärtel M, Reier Ü, Saks Ü, Singh R, Vasar M, Zobel M (2015) Global assessment of arbuscular mycorrhizal fungus diversity reveals very low endemism. Science 349:970–973CrossRefPubMedGoogle Scholar
- Lin K, Limpens E, Zhang Z, Ivanov S, Saunders DGO, Mu D, Pang E, Cao H, Cha H, Lin T, Zhou Q, Shang Y, Li Y, Sharma T, van Velzen R, de Ruijter N, Aanen DK, Win J, Kamoun S, Bisseling T, Geurts R, Huang S (2014) Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus. PLoS Genet 10:e1004078CrossRefPubMedPubMedCentralGoogle Scholar
- Öpik M, Zobel M, Cantero JJ, Davison J, Facelli JM, Hiiesalu I, Jairus T, Kalwij JM, Koorem K, Leal ME, Liira J, Metsis M, Neshataeva V, Paal J, Phosri C, Põlme S, Reier Ü, Saks Ü, Schimann H, Thiéry O, Vasar M, Moora M (2013) Global sampling of plant roots expands the described molecular diversity of arbuscular mycorrhizal fungi. Mycorrhiza 23:411–430CrossRefPubMedGoogle Scholar
- Randle-Boggis RJ, Helgason T, Sapp M, Ashton PD (2016) Evaluating techniques for metagenome annotation using simulated sequence data. FEMS Microbiology Ecology 92:fiw095Google Scholar
- Smith SE, Read DJ (2008) Mycorrhizal symbiosis. Academic Press, AmsterdamGoogle Scholar
- Spatafora JW, Chang Y, Benny GL, Lazarus KL, Smith ME, Berbee ML, Bonito G, Corradi N, Grigoriev I, Gryganskyi A, James TY, O'Donnell K, Roberson RW, Taylor TN, Uehling J, Vilgalys R, White MM, Stajich JE (2016) A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108:1028–1046CrossRefPubMedGoogle Scholar
- Taylor JD, Helgason T, Öpik M (2017) Molecular community ecology of arbuscular mycorrhizal fungi. In: Dighton J, White JF, eds. The fungal community: its organization and role in the ecosystem, 4th edn. CRC Press, 00Google Scholar
- Tedersoo L, Nilsson RH, Abarenkov K, Jairus T, Sadam A, Saar I, Bahram M, Bechem E, Chuyong G, Kõljalg U (2010) 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases. New Phytol 188:291–301CrossRefPubMedGoogle Scholar
- Thiéry O, Vasar M, Jairus T, Davison J, Roux C, Kivistik PA, Metspalu A, Milani L, Saks Ü, Moora M, Zobel M (2016) Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent. Mol Ecol 25:2816–2832CrossRefPubMedGoogle Scholar
- Tisserant E, Malbreil M, Kuo A, Kohler A, Symeonidi A, Balestrini R, Charron P, Duensing N, Frei dit Frey N, Gianinazzi-Pearson V, Gilbert LB, Handa Y, Herr JR, Hijri M, Koul R, Kawaguchi M, Krajinski F, Lammers PJ, Masclaux FG, Murat C, Morin E, Ndikumana S, Pagni M, Petitpierre D, Requena N, Rosikiewicz P, Riley R, Saito K, San Clemente H, Shapiro H, van Tuinen D, Becard G, Bonfante P, Paszkowski U, Shachar-Hill Y, Tuskan GA, JPW Y, Sanders IR, Henrissat B, Rensing SA, Grigoriev IV, Corradi N, Roux C, Martin F (2013) Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis. Proc Natl Acad Sci U S A 110:20117–20122CrossRefPubMedPubMedCentralGoogle Scholar
- Xu TL, Veresoglou SD, Chen YL, Rillig MC, Xiang D, Ondrej D, Hao ZP, Liu L, Deng Y, Hu YJ, Chen WP, Wang JT, He JZ, Chen BD (2016) Plant community, geographic distance and abiotic factors play different roles in predicting AMF biogeography at the regional scale in northern China. Environ Microbiol Rep 8:1048–1057CrossRefPubMedGoogle Scholar