Figure 1.
Outline procedure for development of the duplex PCR-based assay.
These steps were used to measure the levels of bacterial contamination in nucleic acid extracts from cultures of the loricate choanoflagellates Stephanoeca diplocostata and Diaphanoeca grandis.
Figure 2.
Assay Template Composition versus Relative Band Brightnesses for the Linearized Plasmid Mixture Series.
The agarose gel shows the amplification of PCR products from a linearized plasmid-only assay series. The percentages given correspond to the percentage of the 2 µl DNA template volume that was comprised of 4 ng/µl Marinomonas sp. 16S or D. grandis 18S target sequence-containing linearized plasmid. The relative intensity of the relevant band (550 bp for 16S, 400bp for 18S) is observed to vary according to the percentage of target sequence in the template DNA. It should be noted that the percentages quoted concern weight per volume, however the differences in product sizes (400 bp vs. 550 bp) mean that the molar ratios (i.e. number of DNA molecules) are 1.375∶1 18S∶16S by concentration. The 100 bp ladder (Invitrogen) is shown in the first lane with the 1000 bp, 600 bp, 400 bp and 100 bp markers noted.
Figure 3.
Assay Template Composition versus Relative Band Brightnesses for a Simulated Culture Series.
The simulated culture assay series demonstrates that the relationship between assay template composition and relative band brightness still holds even in the presence of background non-target DNA. The agarose gel shows the amplification of PCR products from a simulated culture assay series. The percentages given correspond to the percentage of the 2 µl template DNA that was bacterial Marinomonas sp. gDNA or D. grandis 18S target sequencing-containing linearized plasmid. Both the bacterial gDNA and linearized plasmid were diluted such that the estimated concentration of 16S or 18S target sequence was equal at 0.1 ng/µl. The 100 bp ladder (Invitrogen) is shown in the first lane with the 1000 bp, 600 bp, 400 bp and 100 bp markers noted.
Figure 4.
Assay Standard Curve for use in Quantification of Bacterial Contamination of Loricate Choanoflagellate Cultures.
The relative 16S band brightness is here plotted against percentage of bacterial 16S target sequence in the template DNA. The error bars at each point show ± standard error. This graph can be used as a standard curve to determine the relative levels of bacterial contamination in DNA samples. It may also be used in conjunction with absolute readings of DNA concentration to provide the basis for absolute quantification of the bacterial and choanoflagellate content in the original nucleic acid sample.
Figure 5.
Statistical Verification of Repeatability of Assay Measurements.
Shown are mean relative brightness measurements of the 16S band (bars showing ± standard error) from triplicate assays performed on six monospecific choanoflagellate cultures; three S. diplocostata (S.d. 1–3) and three D. grandis (D.g.1–3). The values are significantly different (F5,57 = 10.48, P<0.001) between the cultures such that measurements are repeatable and representative of the culture they are taken from.
Figure 6.
Assay Measurements from cDNA Template reflect Original Culture Composition.
A The unadjusted cDNA results show a significant difference between gDNA and cDNA results (F5,57 = 13.83, P<0.001). B Applying a 0.149 adjustment to the cDNA results show no significant difference (F5,57 = 1.06, P = 0.403) between gDNA and cDNA results. The bars show mean relative 16S band brightness (±standard error) for either gDNA (grey bars) or cDNA (hatched bars) template from three D. grandis cultures (D.g. X, Y, Z) and three S. diplocostata cultures (S.d. X, Y, Z).
Figure 7.
The dilution series shows a linear relationship between template concentration and relative 16S band brightness for gDNA (A) and cDNA (B). Relative 16S band brightness is plotted against log10 dilution factor. Symbol shape and line style correspond to nucleic acid samples from individual cultures. Grey symbols and lines indicate S. diplocostata, black symbols and lines indicate D. grandis. Trend lines fitted by regression analysis found an average slope of 0.12±0.01 across all samples. Trend lines in all cases had r2 >0.9, with the exception of one dilution set which showed saturation (relative 16S band brightness = 1) after 10-fold dilution (in this case r2 = 0.75).
Figure 8.
The Effect of Antibiotic and Filtration Treatment on the Relative Bacterial Contents of Choanoflagellate Cultures.
White bars are assay results from S. diplocostata, black bars are assay results from D. grandis. For D. grandis the treated residues show a lower bacterial content than the unfiltered and untreated control. For S. diplocostata the treated residue has a slightly higher bacterial content than the control samples. The D. grandis filtrate contains only bacterial signal, whilst for S. diplocostata a relative 18S band brightness of 0.28 is present (X), indicating that choanoflagellates are present in the filtrate.
Table 1.
18S and 16S primer sequences used in the assaying of loricate choanoflagellate cultures.
Table 2.
Recipe for the assay PCR mixture.
Table 3.
Assay PCR protocol.