E.Dogat-Bany,ctal Food Microbiology 85 (2020)103278 iluted 1:5 ere allowed to adsorb to the carbon la oliters P2 nd hal vely filtrated using 0.45 um d again.The grid was im ged at pore)(p 80kV in a Hitac ols P3 and P4) HT7700 transmission electron microscope es.filtrated or not. were then sur 27.Viral DNA extraction After rm in c cing.For one Epo ed with P3.the nd mi d th actioi at 15,000x for 5 min at 4C.the aqucous phase c fre DNA.DNase was inacti d by the addit 2.4.Particles interferometry d organ netrie light mier l-chlo (1:1;saturated w vith 10 mM Tris pH 8. ded tot viral fractio et al (CM nera).We d J (Se Afte adding one volume of phenol-chlorofo The phase w T4 DNA ing that all particles n the te e 50 ul of soc (3M,pH DN at13,000× by20 fold.We next c ed the iM esti (vol/vo of Ep using the Qubit DNA Broad Range assay (ThermoFishe old r give a)from two extr ce). ed at the tion pr II DNA P Kit (Ne of 005 after testi the data ality s)fol 200 cant thre 20 was used with threshold of .05. ibraries with an ave ligat 25.Particles olify the er iodixanol discontinue adients of 45%and 20%(w/v) equimola ed and s d on the mllumina mis m (Mis 10n 36 (Bols e20140 ering 4.5 ml of the 4 vith lengt 25 bp.b of a pipe egy fo as wel d reads (21.1 milion r ads r ss(0.46 million reads per sample in aver 3stage, the supernatant containing free viral particles was diluted 1:5 with cold SM buffer (200 mM NaCl, 10 mM MgSO4, 50 mM Tris pH 7.5) and split, half being used for filtration-free protocols (protocols P1 and P2) and half being successively filtrated using 0.45 μm and 0.2 μm polyethersulfone membranes and glass vacuum filter holders (Milli￾pore) (protocols P3 and P4). Samples, filtrated or not, were then sup￾plemented with 10% PEG 6000 (Sigma) and kept at 4 °C over-night after dissolution for viral particles precipitation. After centrifugation at 6000×g for 1 h at 4 °C, pellets were resuspended with 2 ml of cold SM buffer and split again, half being stocked at 4 °C (protocols P1 and P3) and half being treated with chloroform in order to eliminate membrane vesicles (protocols P2 and P4). More specifically, 1 volume of fresh, non-oxidized chloroform was added to the sample and mixed thor￾oughly for 1 min using a vortex to create an emulsion. After cen￾trifugation at 15,000×g for 5 min at 4 °C, the aqueous phase containing viral particles was recovered. 2.4. Particles counting using interferometry An interferometric light microscope (ILM) (Boccara et al., 2016) was used to count nanoparticles (i.e. both vesicles and viruses) present in our viral fractions as previously described (Roose-Amsaleg et al., 2017). Briefly, 5 μl of each sample were used to collect a stack of 200 images (CMOS camera). We developed a simple ImageJ (Schindelin et al., 2012) script that allows first background subtraction and image quality enhancement, then particles localization on each image. An average number of nanoparticles per frame was obtained. To calibrate the concentration estimates, crude lysates of phages P1, T4, T5, T7, λCI857 and ϕX174 were titrated on Escherichia coli MG1655, and counted with the ILM device. These counts were converted into con￾centrations, assuming that all particles present in the observation vo￾lume (10−8 ml) were detected with the ILM device. Table S1 and Fig. S1 showed a good match between the two measurements, usually within a ± 3 fold range for phages P1, T4, T5, T7 and λCI857, whereas ϕX174 was underestimated by 20 fold. We next compared the ILM estimates with epifluorescence microscopy on two viral samples obtained from Epoisses cheese (Fig. S2). As shown in the result section, virome sam￾ples of Epoisses rind also contain vesicles, which are detected by ILM, but not by epifluorescence, since they mostly do not contain DNA. In accordance with expectations, 1.5–1.9-fold more particles were counted by ILM than by epifluorescence. We concluded that the ILM measure￾ments give reasonable estimates of nanoparticle concentrations (both vesicles and viruses) of viromes. Statistical comparison between ILM measurements (log transformed data) from two extraction procedures was performed using a one-tailed t-test for two dependent means with a significant threshold of 0.05 after testing the data normality using a Shapiro-Wilk test (significant threshold of 0.05). For comparison be￾tween two cheese types, a one-tailed t-test for two independent means was used with a significant threshold of 0.05. 2.5. Particles purity evaluation using iodixanol gradients Two-layer iodixanol discontinuous gradients of 45% and 20% (w/v) in SM Buffer (50 mM Tris pH7.5, 100 mM NaCl, 10 mM MgCl2) were prepared by adding first 6 ml of the 20% fraction in a 12 ml ultra￾centifugation tube, and then under layering 4.5 ml of the 45% fraction in the bottom of the tube, using a glass transfer pipette and a pipette pump. Each viral fraction (1 ml diluted 1:2 with SM Buffer) was finally added on top of the gradient and the tubes were centrifuged at 200,000×g for 5 h at 10 °C, in an SW41 rotor, using a Beckman XL-90 ultracentrifuge. 2.6. Transmission electron microscopy Ten microliters of the viral fraction obtained from an Epoisses cheese with protocol P4 was directly spotted onto a Formwar carbon coated copper grid. Particles were allowed to adsorb to the carbon layer for 5 min and excess of liquid was removed. Ten microliters of a staining uranyl acetate solution (1%) was then spotted to the grid for 10 s and excess of liquid was removed again. The grid was imaged at 80 kV in a Hitachi HT7700 transmission electron microscope. 2.7. Viral DNA extraction The twelve Epoisses samples (three cheeses treated by protocols P1, P2, P3 and P4) were used for viral DNA extraction and shotgun se￾quencing. For one Epoisses sample (Epb) treated with P3, the vesicle band was also recovered from iodixanol gradient for subsequent DNA extraction and sequencing. 500 μl of the viral fraction (or vesicles) were treated for 30 min at 37 °C with 1 U of TURBO DNase (Invitrogen) in order to digest free DNA. DNase was inactivated by the addition of 5.5 μl of 100 mM EDTA and the sample was placed on ice for 5 min. The entire content of the tube was transferred to a 2 ml tube containing a gel that improves separation between the aqueous and organic phases (Phase Lock Gel Heavy; Eppendorf AG, Hamburg, Germany). One vo￾lume of phenol-chloroform (1:1; saturated with 10 mM Tris pH 8.0, and 1 mM EDTA) was added to the sample and, after gentle mixing for 1 min by inversion, the tube was centrifuged at 10,000×g for 10 min at 4 °C.The aqueous phase was then transferred to another Phase Lock Gel tube. After adding one volume of phenol-chloroform and gentle mixing for 1 min by inversion, centrifugation was performed at 10,000×g for 10 min at 4 °C. The aqueous phase was then transferred to another Phase Lock Gel tube. After adding one volume of chloroform and gentle mixing for 1 min by inversion, centrifugation was performed at 10,000×g for 10 min at 4 °C. The aqueous phase (approximately 300 μl) was recovered in a 1.5 ml tube and DNA was precipitated by adding two volumes of absolute ethanol, 50 μl of sodium acetate (3 M, pH 5.2) and 3 μl of glycogen (5 mg/ml; Invitrogen) as carrier. After incubation on ice for 10 min, the DNA was recovered by centrifugation at 13,000×g for 30 min at 4 °C. The DNA pellet was subsequently washed with 800 μl of 70% (vol/vol) ethanol. After centrifugation at 13,000×g for 10 min at 4 °C, the DNA pellet was dried at room temperature for 30 min and dissolved in 20 μl of 10 mM Tris pH 7.5. Purified DNA was quantified using the Qubit DNA Broad Range assay (ThermoFisher Scientific). 2.8. Viral DNA sequencing and bioinformatic analysis Library preparation and sequencing were performed at the GeT￾PlaGe platform (Toulouse, France). Briefly, libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (New England Biolabs) following manufacturer's instructions and using up to 200 ng dsDNA (QuBit quantified, Thermofisher) for each sample, when pos￾sible. DNA was fragmented using covaris M220 (Covaris) leading to libraries with an average insert size of 575 bp. After adapter ligation, 9 PCR cycles were applied to add Illumina adaptators and indexes and to amplify the library. Libraries were then individually quantified by qPCR using the KAPA Library Quantification Kits for NGS (KAPA Biosystems), equimolary pooled and sequenced on the Illumina MiSeq system (MiSeq reagent kit v3, Illumina). Raw reads were quality filtered using Trimmomatic v0.36 (Bolger et al., 2014) in order to remove paired reads with bad qualities (with length < 125 bp, with remnant of Il￾lumina adapters and with average quality values below Q20 on a sliding window of 4 nt). The overall filtering process yielded an average of 2.72 million reads per sample (35.5 million reads in total). The strategy for assembly and mapping is presented in Fig. S3, as well as the number of reads kept at each step. For assembly, only the quality-fil￾tered and paired reads (2 × 1.13 million reads per sample in average, 83% of the total) were kept while those being unpaired at the end of the filtering process (0.46 million reads per sample in average, 17% of the total) were kept aside and used only for mapping. A global assembly was performed using the quality-filtered and paired reads from the E. Dugat-Bony, et al. Food Microbiology 85 (2020) 103278 3