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Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm

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Abstract

A robust and widely applicable method for sampling of aquatic microbial biofilm and further sample processing is presented. The method is based on next-generation sequencing of V4–V5 variable regions of 16S rRNA gene and further statistical analysis of sequencing data, which could be useful not only to investigate taxonomic composition of biofilm bacterial consortia but also to assess aquatic ecosystem health. Five artificial materials commonly used for biofilm growth (glass, stainless steel, aluminum, polypropylene, polyethylene) were tested to determine the one giving most robust and reproducible results. The effect of used sampler material on total microbial composition was not statistically significant; however, the non-plastic materials (glass, metal) gave more stable outputs without irregularities among sample parallels. The bias of the method is assessed with respect to the employment of a non-quantitative step (PCR amplification) to obtain quantitative results (relative abundance of identified taxa). This aspect is often overlooked in ecological and medical studies. We document that sequencing of a mixture of three merged primary PCR reactions for each sample and further evaluation of median values from three technical replicates for each sample enables to overcome this bias and gives robust and repeatable results well distinguishing among sampling localities and seasons.

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Abbreviations

NGS:

Next-generation sequencing

NMDS:

Non-metric multidimensional scaling

DGGE:

Denaturing gradient gel electrophoresis

T-RFLP:

Terminal restriction fragment length polymorphism

FISH:

Fluorescent in situ hybridization

POCIS:

Polar organic chemical integrative sampler

G:

Basic soda lime silicate glass

S:

Stainless steel

A:

Aluminum

PP:

Polypropylene

PE:

High-density polyethylene

STP:

Sewage treatment plant

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Funding

This work was supported by the Czech Science Foundation (No. 15-04258S) and Ministry of Education, Youth and Sports of CR within the LQ1604 National Sustainability Program II (Project BIOCEV-FAR) and by the project “BIOCEV” (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund in the Czech Republic.

The financial support was also given to R. Grabic and V. Zlabek by the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (No. CZ.1.05/2.1.00/01.0024) and CENAKVA II (No. LO1205 under the NPU I program).

Author information

Authors and Affiliations

  1. Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague 4, Czech Republic

    Tomas Bakal, Jiri Janata, Lenka Sabova & Lucie Najmanova

  2. Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in Ceske Budejovice, Vodnany, Czech Republic

    Roman Grabic & Vladimir Zlabek

Authors
  1. Tomas Bakal
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  2. Jiri Janata
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  3. Lenka Sabova
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  4. Roman Grabic
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  5. Vladimir Zlabek
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  6. Lucie Najmanova
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Corresponding author

Correspondence to Lucie Najmanova.

Electronic supplementary materials

Online Resource 1

The list of used composite primers sequences. (XLSX 11 kb)

Online Resource 2

The list of identified taxons (cutoff 0.5%) in experimental and natural samples. (XLSX 59 kb)

Online Resource 3

The number of identified taxons (cutoffs 0.5, 1, 2%) in experimental and natural samples. (XLSX 12 kb)

Online Resource 4

The Bray-Curtis distance matrix calculated for individual experimental and natural samples (cutoff 1%). (XLSX 29 kb)

Online Resource 5

NMDS plot of Bray-Curtis distance matrix for individual experimental samples and medians of natural samples; taxon relative abundance cutoff 0.5% in at least one sample (stress value = 0.149). The values for samples of the experimental set are marked as follows: circle = G; square = A; diamond = PE, triangle = PP; inv. triangle = S. Filled shapes stand for non-rinsed samples, blank ones for rinsed samples. The red star represents the absolute median of all values of the experimental sample set. Natural samples from Zivny stream are marked as follows: dash = upstream STP, cross = downstream STP, gray color = spring, blue = autumn. (PDF 88 kb)

Online Resource 6

NMDS plot of Bray-Curtis distance matrix for individual experimental samples and medians of natural samples; taxon relative abundance cutoff 2% in at least one sample (stress value = 0.137). The values for samples of the experimental set are marked as follows: circle = G; square = A; diamond = PE, triangle = PP; inv. triangle = S. Filled shapes stand for non-rinsed samples, blank ones for rinsed samples. The red star represents the absolute median of all values of the experimental sample set. Natural samples from Zivny stream are marked as follows: Dash = upstream STP, cross = downstream STP, gray color = spring, blue = autumn. (PDF 90 kb)

Online Resource 7

The Bray-Curtis distance matrix calculated for medians from triplicates of experimental and natural samples (cutoff 2%). (XLSX 12 kb)

Online Resource 8

The Bray-Curtis distance matrix calculated for medians from triplicates of experimental and natural samples (cutoff 1%). (XLSX 13 kb)

Online Resource 9

The Bray-Curtis distance matrix calculated for medians from triplicates of experimental and natural samples (cutoff 0.5%). (XLSX 12 kb)

Online Resource 10

The Bray-Curtis distance matrix of comparative samples of medians calculated for triplicates of testing set samples to the absolute median of testing set samples (median calculated for all 30 testing samples) for all three cut-off levels 0.5, 1, and 2% (XLSX 9 kb)

Online Resource 11

The source data for Venn diagrams (Fig. 6). (XLSX 339 kb)

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Bakal, T., Janata, J., Sabova, L. et al. Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm. Folia Microbiol 64, 9–17 (2019). https://doi.org/10.1007/s12223-018-0624-1

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  • DOI: https://doi.org/10.1007/s12223-018-0624-1

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