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Strengths and Biases of High-Throughput Sequencing Data in the Characterization of Freshwater Ciliate Microbiomes

  • Environmental Microbiology
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Abstract

Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of “diversity,” and consequently that neither is entirely superior to the other when investigating environmental protists.

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Acknowledgements

The authors wish to thank Simone Gabrielli for his help in graphical artwork, Valentina Serra and Daniela Carducci for assistance during sampling, and Brad Jones for language editing.

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Author notes

  1. Vittorio Boscaro

    Present address: Department of Botany, University of British Columbia, Vancouver, BC, V6T1Z4, Canada

Authors and Affiliations

  1. Dipartimento di Biologia, Unità di Zoologia-Antropologia, Università di Pisa, 56126, Pisa, Italy

    Vittorio Boscaro, Alessia Rossi, Claudia Vannini, Franco Verni, Sergei I. Fokin & Giulio Petroni

  2. Department of Invertebrate Zoology, St.-Petersburg State University, St.-Petersburg, 199034, Russia

    Sergei I. Fokin

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  1. Vittorio Boscaro
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  2. Alessia Rossi
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Correspondence to Vittorio Boscaro.

Electronic supplementary material

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Online Resource 1

Additional experimental procedures details. (PDF 90 kb)

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Online Resource 2

Number of morphological and Illumina OTUs, divided by ciliate class, in each of the 12 samples. Contig abundances are also reported. (PDF 2444 kb)

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Online Resource 3

Additional pipeline comparisons and statistical test results. (PDF 2468 kb)

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Online Resource 4

Rarefaction curves calculated on the 12 samples. (TIFF 356 kb)

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Online Resource 5

Number of contigs (A) and OTUs (B) assigned to each ciliate subclass in the entire analysis (pipeline U.s.). Some ciliate classes are not subdivided in subclasses [41]; in those cases, we maintained class names (underlined). (TIFF 1167 kb)

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Online Resource 6

Relative number of OTUs per sample, divided by class, according to the morphological analysis (A) and the HTS survey (pipeline U.s.) (B). (JPEG 224 kb)

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Online Resource 7

nMDS analysis performed on presence/absence morphological data from all samples, employing Bray-Curtis distances. Symbols represent the 4 sampling sites according to the legend. The gradient analysis was performed on abiotic factors, represented by vectors. A similar graph including more samples from the same and other sites belonging to the investigated area can be consulted in Rossi et al. [31]. T, temperature; O, oxygen concentration; A, altitude. (EPS 311 kb)

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Boscaro, V., Rossi, A., Vannini, C. et al. Strengths and Biases of High-Throughput Sequencing Data in the Characterization of Freshwater Ciliate Microbiomes. Microb Ecol 73, 865–875 (2017). https://doi.org/10.1007/s00248-016-0912-8

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  • DOI: https://doi.org/10.1007/s00248-016-0912-8

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