Experiment 2: optimizing particle lysis and purification

Viromes were generated as in Experiment 1 with minor changes. First, viruses were resuspended as described for Experiment 1, except half of the samples were not purified with CsCl density gradient centrifugation. This was to follow-up on our previous work that suggested CsCl resulted in potentially a major loss of viruses (Trubl et al., 2016). Second, DNA was extracted from all samples using the PowerSoil method, but the physical method of particle lysis was tested by half of the samples undergoing the standard heat lysis as above and the other half undergoing the alternative PowerSoil bead-beating step (with 0.7 mm garnet beads). Third, the extracted DNA was further cleaned up with DNeasy PowerClean Pro Cleanup Kit (Qiagen, Hilden, Germany, product 12997), instead of AMPure beads. Assessment of microbial contamination was done via qPCR (pre and post-cleanup) with primer sets 1406f (5′-GYACWCACCGCCCGT-3′) and 1525r (5′-AAGGAGGTGWTCCARCC-3′) on 5 µl of sample input to amplify bacterial and archaeal 16S rRNA genes as previously described (Woodcroft et al., 2018). Finally, the 12 palsa samples were sequenced at the Joint Genome Institute (JGI; Walnut creek, CA), where library preparation was performed using the Accel-NGS 1S Plus kit. All viromes required 20 PCR cycles, except –CsCl, bead-beating which required 18. All libraries were sequenced using the Illumina HiSeq-2000 1TB platform (2 × 151 bp paired-end).