Cell strains and growth conditions
Cultures of Euglena gracilis Z (SAG 1224-5/25), Euglena hiemalis (CCAP 1224/35), and Euglena longa (CCAP 1204-17a) were cultivated statically in the Cramer-Myers medium (Cramer and Myers 1952), supplemented with ethanol (0.8% v/v) and aqueous soil extract (1% v/v). Cells were grown at 18 °C under white light exposure (16:8-h light/dark cycle, ca. 27 μmol photons m−2 s−1).
Disc diffusion antibiotic sensitivity testing
In order to determine the antibiotic susceptibility of contaminant organisms, the agar diffusion test was performed (Bauer et al. 1966). The initial, non-axenic cultures of euglenids were plated on Tryptone Soya Yeast Extract Agar (TSYEA; BTL) supplemented with amphotericin B (1% v/w; Sigma). Then, the antibiotic-impregnated paper discs (Oxoid) were placed on the plates and left to incubate. Our previous experience has shown that antibiotics affecting DNA or protein synthesis, particularly those recently developed, are more lethal for euglenids at lower concentrations than for their bacterial and/or fungal contaminants. Therefore, they were not taken into account in this study. Various agents inhibiting bacterial cell wall synthesis, such as ampicillin (25 μg), cefotaxime (30 μg), fosfomycin (50 μg), gentamicin (30 μg), penicillin (25 μg), rifampicin (30 μg), trimethoprim (2.5 μg), and vancomycin (30 μg), were used in the antibiotic screening, as the least harmful for the cells of euglenids. Cefotaxime and vancomycin—the compounds generating the largest zones of inhibition—were chosen for the final purification procedure.
The initial cultures of euglenids were mechanically pre-purified by centrifugation (2500×g, 30 s, RT) and washing with distilled water (each time the supernatant was discarded). The procedure was repeated as long as the amount of bacteria observed under the microscope was visibly decreasing. Such prepared cultures were diluted and streaked on solid Cramer-Myers medium supplemented with mineral medium (5% v/v) (Starr 1964), aqueous soil extract (5% v/v), and amphotericin B (1% v/w; Sigma), and agarised with TSYEA (1% v/w; BTL). In order to obtain zones with decreasing concentrations of antibiotics, only two discs, one for each of the selected compounds (cefotaxime and vancomycin, respectively), were placed on the opposite sides of the plate (supplementary Figure S1, supplementary material online). Grown Euglena colonies (visible under the microscope as bacteria-free and alive) were subsequently restreaked on the same medium for further purification. To increase survivability of the Euglena cells, the antibiotic discs were placed on the plates every second passage. The procedure was repeated until the axenic algal cultures were obtained. Afterwards, they were transferred to the liquid medium and constantly monitored for bacterial and fungal presence.
Genomic DNA extraction protocols
Five DNA extraction methods were evaluated in this study. The DNA was isolated from all three species (E. gracilis, E. longa, E. hiemalis) in pentaplicates with each extraction protocol. The initial experimental steps remained the same in all cases. A total volume of 10 mL of liquid cultures in the logarithmic growth phase was centrifuged (5000×g, 5 min, RT) and rinsed with nuclease-free water three times to completely remove the residues of the growth medium. Washed Euglena cells were then resuspended, aliquoted (1 mL), and centrifuged. Then, each of the cell pellets (± 50 mg) was processed in accordance with the chosen method’s requirements. Finally, the DNA was eluted or resuspended in 100 μL of nuclease-free water (GE Healthcare).
Extraction with commercial silica-membrane column kits
Two commercially manufactured kits, designed for quick purification of genomic DNA—DNeasy Blood & Tissue (Qiagen) and DNeasy Plant (Qiagen)—were tested. In the case of the DNeasy Blood & Tissue kit, the spin column protocol designed for purification of total DNA from animal blood/cultured cells was applied, whereas in the case of DNeasy Plant kit, the TissueRuptor protocol with liquid nitrogen was used. All steps were performed strictly as described in the instructions provided by the manufacturer. In both cases, on-column RNAse A (100 mg mL−1, 4 μL, 2 min, RT; Qiagen) digestion was carried out.
Extraction with phenol:chloroform
Genomic DNA was isolated with standard ethanol precipitation following phenol:chloroform:isoamyl alcohol (24:25:1 v/v; AppliChem) treatment, according to the previously described, albeit slightly modified protocol (Psifidi et al. 2010; Green and Sambrook 2017). Specifically, 1 mL of lysis buffer containing 10 mM Tris HCl (pH = 7.5), 1 mM EDTA, 50 mM NaCl, 0.2% SDS (v/w), and 1 mg of proteinase K (Qiagen) was used to digest Euglena cells for 1.5 h at 56 °C. The lysate extraction was performed twice with 1 mL of phenol:chloroform:isoamyl alcohol (24:25:1 v/v; AppliChem). Following the first extraction step, the aqueous phase was treated with RNAse A (100 mg mL−1, 4 μL; Qiagen) and incubated at 37 °C for 30 min with periodic, gentle mixing. Afterwards, the extraction was repeated. Then, 2.5 volume of absolute ethanol (AppliChem) and 0.1 volume of 3 M sodium acetate (pH = 5.2) were added and the DNA was precipitated at − 20 °C for 1.5 h. The sample was spun at 12,000×g, 10 min, RT, and the DNA pellet was washed twice with 70% ethanol. Finally, the pellet was air-dried and resuspended in nuclease-free water.
Extraction with traditional CTAB method
The cetyltrimethylammonium bromide (CTAB; AppliChem) DNA isolation was performed strictly as described elsewhere (Allen et al. 2006). The volumes of utilized reagents were downscaled according to the amount of the initial Euglena biomass.
Extraction with modified CTAB method
The CTAB-based, rapid DNA extraction protocol (Healey et al. 2014) was slightly modified. Prior to the proper isolation, the cell pellets were washed with ice-cold DMSO:acetonitrile (1:1 v/v; AppliChem). This step was introduced to perforate the pellicle (and to facilitate the penetration of the lysis buffer into the cells), as well as to remove the photosynthetic pigments, polysaccharides, and wax esters which hinder nucleic acids isolation and purification (Rosenberg 1967; Mederic et al. 1987; Barsanti et al. 2001; del Campo et al. 2010; Healey et al. 2014). Also, the RNAse A (100 mg mL−1, 4 μL; Qiagen) treatment was extended to 30 min in comparison to the guidelines provided by Healey et al. (2014).
DNA integrity assessment
The integrity of the DNA samples obtained using each tested extraction method was examined through standard gel electrophoresis (Psifidi et al. 2015). In detail, 5 μL of each DNA extract was analyzed in a 1.5% agarose gel stained with 0.5% Midori Green (Nippon), run in 1× TAE buffer. DNA bands were visualized using the ChemiDoc UV transilluminator (Bio-Rad).
DNA purity and yield
For each of the applied extraction procedures, the concentration and purity of the recovered DNA were assessed spectrophotometrically with NanoPhotometer NP80 (Implen). An Abs260/280 ratio was used to evaluate protein contamination while an Abs260/230 ratio was used to determine organic solvents contamination. Afterwards, for each species/isolation method, one sample (with the best parameters) was selected based on the absorbance values and subjected to fluorimetric measurements. Concentration of the DNA in those samples was further examined using the High Sensitivity DNA Assay implemented by Qubit 3.0 fluorometer (Thermo Scientific). Each time, 1.5 μL (absorbance) or 1 μL (fluorescence) of DNA sample (or nuclease-free water as a blank solution) was used during the sample assessment. Each measurement was performed twice, and obtained values were averaged. Total DNA yield was calculated based on DNA concentration derived from the NanoPhotometer and Qubit results calculated together with the total volume of the DNA extract.
Application of the isolated DNA in high throughput sequencing
Based on the average values of the above parameters, out of five extraction methods tested, the most effective and robust one was selected. In order to evaluate its application in NGS library construction and sequencing, single isolates of E. hiemalis and E. longa, which exhibited optimal parameters of concentration and quality, were chosen for further manipulations. The DNA prepared for sequencing was stored in − 20 °C no longer than a few days, avoiding its exposure to temperature amplitudes.
Preparation of a pair-end reads (PE150) library was carried out externally using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB) and sequenced commercially on an HiSeq4000 instrument (Genomed, Warsaw, Poland). The quality of the DNA library was assessed using a 2100 Bioanalyzer (Agilent). Additionally, the quality of raw sequencing reads after trimming (removal of library adaptors) was analyzed using the FastQC software (Andrews 2010).