Methods |
Corresponding author: Martyn G. Kelly ( mgkelly@bowburn-consultancy.co.uk ) Academic editor: Emre Keskin
© 2021 Martyn G. Kelly, Tim Jones, Kerry Walsh.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kelly MG, Jones T, Walsh K (2021) Potential for cross-contamination of diatom DNA samples when using toothbrushes. Metabarcoding and Metagenomics 5: e66503. https://doi.org/10.3897/mbmg.5.66503
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The use of toothbrushes and similar devices for sampling diatoms from hard surfaces is a well-established approach. Toothbrushes are routinely cleaned and reused when sampling for analysis by light microscopy. This paper looks at the scale of contamination encountered when this technique is used to sample diatoms for metabarcoding analyses, as well as at the scale of contamination to be expected if stream, rather than distilled water, is used to wash diatoms from stones. Although some contamination attributable to toothbrushes was detected, read numbers were low and had no effect on index calculation or ecological status estimates. However, if the primary focus of a study is to thoroughly document diversity in a sample, then even this small level of contamination may be unacceptable and more stringent measures may be required.
diatoms, metabarcoding, ecological assessment, Water Framework Directive
Although DNA-based technologies have potential to improve ecological assessment (
The standard method for sampling diatoms for ecological status and water quality assessment in Europe and beyond is to brush or scrape the upper surface of a hard substratum (
This study investigates the scale of contamination introduced by the reuse of toothbrushes at different sites and on the effect of using stream water, rather than pure water, to rinse biofilms from surfaces. It has the same basic design as the study by
Details of the sites are given in Table
Background information on the sites used in the study. Values for chemical variables are medians (minimum – maximum) from the closest water chemistry sampling point for the 12 months before March 2017 (R. Nadder: 5 samples) and for 2015, the most recent data for Ober Water prior to sampling (6 samples).
Nadder, Tisbury Station | Ober Water, upstream A35 | |
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Location: | ||
UK National Grid Reference | ST 94616 29152 | SU 24964 03815 |
Latitude/Longitude | 51°03’N, 002°04’W | 50°50’N, 001°38’W |
Altitude (m) | 90 | 30 |
Water chemistry: | ||
Alkalinity (mgl-1 CaCO3) | 186 (82–205) | 16 (10–20) |
pH | 8.0 (7.8–8.2) | 8.1 (7.5–8.6) |
Conductivity (µScm-1) | 512 (246–527) | 142 (120–147) |
Ammonia-N (mgl-1) | 0.042 (0.03–0.157) | < 0.03–0.063 |
Nitrate-N (mgl-1) | 4.12 (1.98–4.45) | < 0.196–0.246 |
Reactive P (mgl-1) | 0.169 | 0.0047 (0.0031–0.0068)5 |
Current ecological status: | ||
Overall | Moderate | Good |
Macrophytes and phytobenthos | Moderate | High |
Phosphorus | Moderate | High |
Five samples were collected at each site for each of the following three treatments:
• brand new toothbrushes using distilled water to wash the biofilm into sample bottles;
• brand new toothbrushes using stream water from site to wash the biofilm into sample bottles; and
• toothbrushes previously used at the other site, along with stream water from the sampling site
In addition, three control samples (5 ml each) were collected:
• one using just distilled water; and,
• one each using river water from the two sites.
Sampling involved brushing the upper surface of five cobble-sized stones and collecting the suspension in a tray. Using a new, but non-sterile Pasteur pipette, 5 ml of the suspension of biofilm and water was transferred to a sterile 15 ml centrifuge tube containing 5 ml nucleic acid preservative, consisting of 3.5 M ammonium sulphate, 17 mM sodium citrate and 13 mM ethylenediaminetetraacetic acid (EDTA). Samples were then transferred to the laboratory in a cool box and frozen at -30 °C prior to DNA extraction. The methods used for DNA extraction, amplification and analysis followed methods described in
Non-metric multidimensional scaling (NMDS:
The Trophic Diatom Index (TDI5NGS) was calculated following
The distilled water control sample contained just 23 reads compared with an average of 27,181 reads for all other samples. This control sample is not considered further.
The diatom assemblage from the River Nadder in samples collected with unused toothbrushes was dominated by Navicula lanceolata (average relative abundance: 41%) along with Amphora pediculus (8%), Melosira varians (8%), Nitzschia recta (7%) and Navicula gregaria (5%). In contrast, the diatom assemblage from Ober Water was dominated by Achnanthidium minutissimum (38%), along with Platessa oblongella (15%) and Gomphonema truncatum (11%).
Non-metric multidimensional scaling (NMDS) of the dataset yielded an ordination with very low stress (0.0684), with a clear separation between the two sites along axis 1 (Figure
Plot showing position of samples from River Nadder and Ober Water relative to the first 2 axes of an NMDS ordination.
If there is a significant amount of contamination, then taxa that are abundant at one site should be present in raised numbers in samples collected using dirty equipment at the other site, but rare in the others. Although significant effects were observed for several taxa, the scale of the effect was generally small, particularly for samples from the River Nadder where the increased representation in samples collected with contaminated toothbrushes exceeded 1% only for Achnanthidium minutissimum (Figure
Variation in relative abundance of taxa carried over from one site to the other, using a clean toothbrush and one used previously at the other site.
A similar approach was adopted to look at possible contamination from stream water. The relative abundance of the most abundant taxa in the stream water sample from each stream was compared with the samples washed with stream and distilled water from that location.
In the case of the River Nadder, the stream water was dominated by planktonic diatoms (65% of total reads). Three of these – Stephanodiscus hantzschii, Cyclostephanos invisitatus and Discotella sp. – were all elevated with respect to the distilled water sample (Figure
Variation in proportions of taxa that were abundant in stream water from the River Nadder at the time of sampling in biofilm samples collected with stream and distilled water, respectively.
There were almost no planktonic diatoms in the Ober Water stream water; however, the composition of the sample was quite different to that of biofilm samples, with a greater proportion of nutrient-rich taxa. There was, despite this, no significant increase in proportions of these taxa in the biofilm when stream water was used to wash the stones (Figure
Variation in proportions of taxa that were abundant in stream water from Ober Water at the time of sampling in biofilm samples collected with distilled and river water, respectively.
There is no significant difference between treatments when TDI5NGS scores are calculated on samples from the River Nadder (Kruskal-Wallis test: p = 0.14). By contrast (and counterintuitively), TDI5NGS is significantly lower in samples from Ober Water which were removed with toothbrushes formerly used in the more enriched River Nadder (Figure
The results of this study highlight a potential for toothbrushes to retain traces of the diatom assemblage (and, presumably, other constituents of stream biofilms), even after the routine cleaning procedure (washing bristles vigorously in the stream and rubbing against waders:
Contamination from the stream water used to wash the samples appears to be less of a problem. In the case of the River Nadder, planktonic taxa dominated the suspended diatom assemblage. Planktonic taxa do not contribute to the TDI5NGS score and so should have no effect on the final index value, However, several of these have large cells with multiple chloroplasts and there may be issues when sampling coincides with a plankton bloom (see
However, our results also show that contamination, both from dirty equipment and upstream sources of DNA, do influence the composition of assemblages and, therefore, it is reasonable to assume that they may affect the final assessment in a few cases. Earlier studies, using morphological identification by light microscopy, had found variation of up to 7 TDI units between replicate samples collected on the same day (
This research was funded by the Environment Agency of England: Project number SC160014.