Dataset: Asteroid viral and bacterial metagenome genetic accession information
Deployment: SeaStarMicrobiology_Hewson

For viral metagenomes:

1 g of preserved or frozen tissue was homogenized in 25 mL of 0.02um filtered PBS in a NutriBullet blender. The homogenate was centrifuged briefly at 3,000 x g to remove large tissue debris and ossicles, and the supernatant filtered through 0.2um Durapore filters to remove cellular debris. The filtrate was precipitated with PEG-8000 (1 g/ml) overnight. The filtrate was then centrifuged at 22,000 x g for 30 min, the supernatant decanted, and the pellet resuspended in 1 ml 0.02 um filtered PBS. The resuspension was filtered through a 0.2 µm Acrodisc PES filter. Samples were then treated with DNAseI, RNAse One and Benzonase for 3 h. Nuclease activity was stopped by the addition of x vol ETDA (25 mM). Viral DNA in the purified suspension was extracted using the Zymo Viral DNA kit. The DNA was amplified prior to sequencing using the WGA2 kit V2 (GE Biosciences) before submission to the Cornell Biotechnology Resource Center (BRC) for library preparation and sequencing.

For bacterial metagenomes:

Samples of asymptomatic asteroid tissues (~ 1cm2) were excised from dorsal surfaces and lesioned tissues were excised from symptomatic asteroids, and then homogenized in 10mL of 0.02 µm filtered phosphate buffered saline using the NutriBullet for 1 minute. Homogenates were allowed to settle for 5 min before 1 mL supernatant was removed and placed into sterile microcentrifuge tubes. The homogenate (400 µL) was purified on PERCOLL step gradients (800 µL each step at 70% and 30%) to remove host cells. Gradients were centrifuged at 12,000 x g for 90 min (4oC). The interface between 30% and 70% was determined with reference to a dense E. coli culture run on an identical gradient. The interface was removed and DNA extracted using the ZR Bacterial & Fungal DNA kit (Zymo Research) following manufacturer’s recommendations. The interface fraction was then targeted for metagenomic library preparation. DNA was extracted from the cell fraction and amplified using the GenomePLEX kit (Sigma Aldrich).

Collection note: The sea star samples were collected by collaborators (samples other than P. ochraceus or P. helianthoides - these were collected by Ian Hewson) intertidally by hand.