Viral metagenome prepared from Daphnia mendotae tissues
Viruses associated with Daphnia tissues were processed according to protocols of Thurber et al., 2009. Approximately half of all washed Daphnia from July and August were first thawed, supplemented with 10 ml 0.02 µm-filtered phosphate buffered saline (PBS), and ground in a sterile mortar and pestle for 5 min. The homogenate was then syringe-filtered through 0.2 µm Acrodiscs (Millipore) to remove cell debris and particles. The filtrate was amended to 32 ml with 0.02 µm PBS and 10% PEG-8000 (weight/volume), shaken, then allowed to precipitate overnight at 4oC. Samples were then centrifuged at 22,000 x g for 1 h and supernatant decanted. Viral pellets were resuspended in 3 ml of 0.02 µm filtered PBS. The filtrate was subject to density gradient ultracentrifugation, using density steps at 1.3, 1.5 and 1.7 g mL-1 and performed in a swing-bucket rotor at 60,000 x g for 2 h. Viruses in the 1.5 – 1.7 g mL-1 fraction (~ 1 mL) were removed using a sterile syringe, and re-filtered through a 0.2 µm PES filter. The concentrate was treated with nucleases (2.5U of DNAse and 0.25U RNAse) for 2 h at 37oC to eliminate nucleic acids not incorporated into viral capsids (Ng et al. 2009a). DNA was extracted from 500 µl of each using the CTAB/EDTA extraction procedure outlined in (Thurber et al. 2009). Extracted DNA was tested for the presence of cellular DNA by amplification of both 16S rRNA and 18S rRNA genes following protocols of (Thurber et al. 2009). In all samples, there were no amplicons generated after 30 cycles of PCR, indicating cell lysates were clear of contaminating cellular DNA.    Daphnia viral shotgun libraries were prepared for each sample using Genomiphi ©, where 1 µl of purified viral DNA was used as template material. Five Genomiphi reactions were pooled and purified using the DNEasy Tissue kit (Qiagen). Cleaned Genomiphi reactions were quantified by Pico Green fluorescence, and submitted for sequencing at EnGenCore (University of South Carolina), where each sample was run on 1/8th of a picotiter plate using Titanium pyrosequencing chemistry.