Viral metagenome prepared from Diporeia spp. tissues
Diporeia spp. samples (10 individuals from each lake) from Owasco Lake, Lake Superior, and Lake Michigan were thawed and immediately ground in 10 mL virus-free (0.02 µm filtered) phosphate buffered saline (PBS) using a mortar and pestle. The homogenate was subject to metaviromic preparation according to (Ng et al., 2010). Amphipod homogenate was filtered through 0.2 µm PES syringe filters, then viruses were precipitated by the addition of PEG-8000 (10% weight/volume) overnight at 4oC (Thurber et al., 2009). After precipitation, the samples were centrifuged at 15,000 x g and supernatant discarded. Pellets containing viral particles were resuspended in 1 mL of 0.02 µm-filtered nuclease-free water, filtered through 0.2 µm syringe filters, then transferred to sterile microcentrifuge tubes. The viral suspension was treated with RNAse A (20 U) and DNAse I (10 U) at 37oC for 2 hr to remove DNA from the suspension that was not in capsids. Enzyme activities were slowed by the addition of 100 mM EDTA. The presence of virus particles (and absence of bacterial cells) was checked by subjecting a 50 µL aliquot of the virus preparation to SYBR Green I epifluorescence microscopy (Noble and Fuhrman, 1998). Viral DNA was extracted using the DNEasy tissue kit with modifications according to (Thurber et al., 2009). The DNA was checked for the presence of bacterial and eukaryotic genome fragments by PCR of 16S and 18S rRNA genes (Thurber et al., 2009), which were negative. Extracted DNA (1 µL; quantification was unsuccessful as DNA was lower than detection threshold) was amplified by multiple displacement amplification using the Genomiphi V2 kit. Five reactions of each sample were pooled into a single tube and DNA purified using the DNEasy kit (Thurber et al., 2009). Five µg of amplified DNA from each location was subject to Illumina paired-end (200 bp) sequencing at the Cornell University Core Laboratories Center. Prior to sequencing, DNA was randomly sheared to 400 – 600 bp, ligated to adapters and bar-coded with Illumina hexamers.