Supplementary material: Choice of pore size can introduce artefacts when filtering picoeukaryotes for molecular biodiversity studies
Nikolaj Sørensen ∙ Niels Daugbjerg ∙Katherine Richardson
N. Sørensen () ∙ K. Richardson
Center for Macroecology, Evolution and Climate
University of Copenhagen
Universitetsparken 15
2100 Copenhagen, Denmark
e-mail:
N. Sørensen ∙ N. Daubjerg
Marine Biological Section
Department of Biology
University of Copenhagen
Universitetsparken 4
2100 Copenhagen, Denmark
Author for correspondence:
Methods
Primer design
For designing dinoflagellate-specific primers, an alignment of partial 18S rDNA sequences consisting of 13 target-dinoflagellate and 60 non-target phylotypes, mainly novel alveolate I and II which are closely related to core dinoflagellates (Guillou et al. 2008), was visually inspected for suitable primer sites. Putative primer pairs were then analysed in CLC Main Workbench 6.6.2 (CLC bio) and by a NCBI BLAST search (Altschul et al. 1990). The primers CDF670f (5´-GCATCYTCTTGGWGAACG-3´) and CDF1058r (5´-GTGCTGAAGGAGTCGT-3´) were designed. This is a dinoflagellate-specific primer pair, which amplifies approximately 350 bp of the 18S rDNA. The numbering is based on the 18S rDNA sequence of Prorocentrum micans (AJ415519).
For assessing the specificity of the designed CDF670f-CDF1058r primer pair, they were tested on 52 plasmids from recombinant clones containing environmental 18S rDNA from a previous study on picoeukaryotic diversity (Sørensen et al. 2011). Six of these were positive controls (dinoflagellate), while the remaining 46 were negative controls (Table S3). As circular plasmids show significantly decreased amplification by PCR (Hou et al. 2010), all plasmids were digested with the restriction enzyme NotI by mixing 2.5 µL plasmid, 6.5 µL H2O, 1 µL 10x buffer and 0.5 µL 10,000 U/mL NotI and incubating it at 37 °C for 1 h. The restriction enzyme was subsequently heat inactivated at 65 °C for 15 min. NotI was used as it recognizes very few restriction sites, making fragmentation unlikely. Based on the sequence of the plasmid vector (pJET1.2/blunt, Fermentas) and the inserts (Sørensen et al. 2012), only a single restriction site could be found in all clones, which was placed in the vector.
Results
Primer design
In Table S3,the average Cq-value for positive and negative controls were 11.10 and 33.85 respectively, equivalent to a 218,772-fold difference in starting quantities (assuming an amplification efficiency of 71.7%, see below), when excluding two outliers. In addition,17 of the negative controls showed no amplification. The lowest difference in Cq-value between a positive and negative control was 11.00. The two outliers were one of the 46 negative controls, which showed high amplification with a Cq-value of 18.11 (300709_06 belonging to a putative sister group to Dinokaryota, (Sørensen et al. 2011) and has only one mismatch to CDF1058r) and one of the 6 positive controls, which showed low amplification with a Cq-value of 29.82 (100609_39, that has 5 mismatches with CDF1058r near the 3´-end).
The primer pair showed an amplification efficiency of 71.7% on the linearised plasmid that was used as the standard (010609_08). The efficiency was higher at lower annealing temperatures but this, in turn, also increased the amplification of negative controls (data not shown). The melting curve showed a single peak at 77.0-77.5 °C in all samples.
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ(1990) Basic Local Alignment Search ToolJ Mol Biol215:403-410
Guillou L, Viprey M, Chambouvet A(2008) Widespread occurrence and genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata) Environ Microbiol 10:3349-3365
Hou Y, Zhang H, Miranda L, Lin S(2010) Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene PLoS One 5:e9545
Sørensen N, Daugbjerg N, Gabrielsen TM(2012) Molecular diversity and temporal variation of picoeukaryotes in two Arctic fjords, Svalbard Polar Biol 35:519-533
Table S1 Relative abundance of dinoflagellates, ciliates and rhizarians in published molecular picoeukaryotic studies and the pore size of the end filterEndfilter / Dinoflagellates / Ciliates / Rhizarians / Protist clones/tagsa / Reference
0.1 µm / 6.9% / 4.3% / 18.1% / 116 / 34e
0.2 µm / 12.9% / 14.9% / 10.1% / 248 / 17
0.2 µm / 2.0% / 2.0% / 1.5% / 200 / 25c
0.2 µm / 14.7% / 2.3% / 13.7% / 511 / 32
0.2 µm / 31.8% / 4.3% / 9.1% / 484 / 38
0.22 µm / 21.3% / 0.7% / 31.2% / 143,085 / 1
0.22 µm / 6.4% / 5.0% / 2.3% / 220 / 9
0.22 µm / 2.7% / 5.6% / 9.3% / 409 / 37
0.45 µm / 3.7% / 3.7% / 7.4% / 108 / 21b
0.45 µm / 4.0% / 0.0% / 20.9% / 225 / 31
0.45 µm / 3.8% / 12.9% / 7.1% / 364 / 36
0.45 µm / 2.3% / 4.7% / 11.6% / 43 / 40f
0.6 µm / 4.8% / 0.0% / 38.7% / 62 / 34
0.6 µm / 0.9% / 0.9% / 2.7% / 111 / 34d
0.8 µm / 2.1% / 0.0% / 0.0% / 47 / 34
aAll eukayotic sequences found, excluding metazoans and fungi
bSequenced extracted bands from DGGE TTGE
cUnamended incubation experiment after 6 or 8 days
dBased on 18S rRNA sequences
eThe sum of the 0.1-0.8 and 0.8-3 µm size fractions
f Dataare based on number of fully sequenced clones
Table S2 Relative abundance of dinoflagellates, ciliates and rhizarians in molecularpicoeukaryotic studies using GF/F as the end filter
Dinoflagellates / Ciliates / Rhizarians / Protist clones/tagsa / Reference
19.2% / 11.1% / 5.5% / 109,212 / 3
7.3% / 17.4% / 4.8% / 729 / 4
5.6% / 0.0% / 8.3% / 36 / 29
aAll eukaryotic sequences found, excluding metazoans and fungi
Table S3 Amplification of negative and positive controls for the
dinoflagellate specific primer pair CDF670f-CDF1058ra
Clone name / Accession number / Taxon / Cq
010609_08 / HQ156810 / Dinoflagellate / 15.23
020609_01 / HQ156813 / Dinoflagellate / 12.62
080609_04 / HQ156819 / Dinoflagellate / 12.18
100609_39 / HQ156844 / Dinoflagellate / 29.82
230609_08 / HQ156887 / Dinoflagellate / 8.19
300709_09 / HQ156893 / Dinoflagellate / 7.3
190609_12 / HQ156874 / Clade A / N/A
300709_06 / HQ156892 / Clade A / 18.11
080609_01 / HQ156818 / Novel alveolate II / N/A
080609_18 / HQ156823 / Novel alveolate II / N/A
100609_05 / HQ156825 / Novel alveolate II / 35.06
100609_07 / HQ156826 / Novel alveolate II / 33.27
100609_14 / HQ156831 / Novel alveolate II / 36.91
100609_24 / HQ156836 / Novel alveolate II / 31.62
100609_38 / HQ156843 / Novel alveolate II / N/A
130609_08 / HQ156851 / Novel alveolate II / N/A
130609_12 / HQ156853 / Novel alveolate II / N/A
170609_10 / HQ156865 / Novel alveolate II / N/A
170609_12 / HQ156866 / Novel alveolate II / N/A
210609_04 / HQ156875 / Novel alveolate II / N/A
210609_05 / HQ156876 / Novel alveolate II / 35.45
300509_08 / HQ156888 / Novel alveolate II / N/A
300509_11 / HQ156889 / Novel alveolate II / 36.24
300709_01 / HQ156891 / Novel alveolate II / 36.64
300709_12 / HQ156896 / Novel alveolate II / N/A
040609_01 / HQ156817 / Novel alveolate I / 35.08
080609_06 / HQ156820 / Novel alveolate I / N/A
100609_13 / HQ156830 / Novel alveolate I / N/A
100609_48 / HQ156846 / Novel alveolate I / 36.84
130609_03 / HQ156849 / Novel alveolate I / 34.59
150609_01 / HQ156859 / Novel alveolate I / 35.6
170609_09 / HQ156864 / Novel alveolate I / 36.35
210609_12 / HQ156878 / Novel alveolate I / 28.57
300509_13 / HQ156890 / Novel alveolate I / 34.94
100609_10 / HQ156827 / Ciliate / 37.97
100609_29 / HQ156838 / Ciliate / N/A
170609_08 / HQ156863 / Ciliate / N/A
170609_14 / HQ156867 / Ciliate / 34.97
170609_15 / HQ156868 / Ciliate / 25.45
210609_17 / HQ156880 / Ciliate / 34.17
230609_02 / HQ156882 / Ciliate / N/A
230609_05 / HQ156885 / Ciliate / 36.6
150609_04 / HQ156861 / Rhizaria / 39.25
170609_19 / HQ156870 / Rhizaria / N/A
100609_40 / HQ156845 / Picobiliphyte / 33.55
100609_22 / HQ156858 / MAST-1A / 26.89
130609_13 / HQ156854 / MAST-1B / 33
100609_12 / HQ156894 / Crysophyceae / 33.66
100609_23 / HQ156835 / Micromonas pusilla / 26.41
230609_07 / HQ156886 / Choanoflagellida / 26.23
170609_17 / HQ156869 / Haptophyceae / 33.42
170609_04 / HQ156862 / Cryptophyceae / 32.1
aN/A: the plasmid showed no detectable amplification (Cq > 60)