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

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  • Martti Vasar
  • Reidar Andreson
  • John Davison
  • Teele Jairus
  • Mari Moora
  • Maido Remm
  • J. P.W. Young
  • Martin Zobel
  • Maarja Öpik


Publication details

DateAccepted/In press - 12 Jul 2017
DateE-pub ahead of print - 20 Jul 2017
DatePublished (current) - 1 Nov 2017
Issue number8
Number of pages13
Pages (from-to)761-773
Early online date20/07/17
Original languageEnglish


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.

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© 2017 Springer-Verlag GmbH Germany. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

    Research areas

  • 454 Sequencing, Arbuscular mycorrhiza, Community diversity, Illumina sequencing

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