Increased sequencing depth does not increase captured diversity of arbuscular mycorrhizal fungi
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.
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