Dataset: Viral metagenomic investigation of the Caribbean echinoderm Holothuria floridana collected in Marathon, Florida on 25 Aug 2023

Specimens (n = 4) of grossly normal Holothuria floridana (urn:lsid:marinespecies.org:taxname:210900) were collected offshore of Marathon, Florida (24.7592N, 81.1076W) on 2023-08-25 and immediately subsampled by five mm biopsy punches, which were transferred to sterile cryovials per Crandell et al. (2023). Punches were frozen at −80 °C on arrival to the lab at the Florida Fish and Wildlife Commission (within an hour of collection) before shipping to Cornell University. Small subsamples of each specimen were prepared for viral metagenomic sequencing broadly following the approach of Ng et al. (2011). Briefly, tissues were homogenized in sterile, 0.02 µm filtered phosphate buffered saline by beating using Zymo ZR Basher Beads (part number S6012-50) in a Biospec Instruments homogenizer for 1 min at maximum speed. The homogenates were centrifuged at 3,000 × g for 1 min to remove large cell debris, before filtering the supernatant through 0.2 µm pore size polyethersulfone syringe filters. Filtered homogenates were treated with 5U Turbo DNase (Invitrogen; part number AM2238), 5U Benzonase nuclease (Sigma Aldrich; part number E1014) and 20U RNase ONE (Promega; part number M4261) for 2 hours at 37 °C to digest extracellular and non-ribosome-bound nucleic acids. Following incubation, RNA was extracted from subsamples of purified virus-sized material using the Zymo Viral RNA kit (part number R1034) before storing RNA at −80 °C until further processing.

RNA in viral extracts was amplified using the SeqPlex RNA kit (Sigma Aldrich; part number SEQXE-10RXN, n = 11 libraries). Subsequent specimens were amplified using the TransPlex kit (WTA2; Sigma Aldrich; part number WTA2-10RXN, n = 3 libraries), which was used in prior RNA viral metagenomic work. Amplification products were purified using the Zymo DNA Clean and Concentrator -5 (part number D4004), quantified by Pico Green fluorescence (Invitrogen; part number P11496), and submitted for sequencing at the Cornell Biotechnology Resource Center. Libraries were prepared for sequencing using the Nextera Flex kit (Illumina; part number 20018704) and sequenced on the Illumina MiSeq platform using the 500 bp Nano kit. Sequences from all libraries were archived at NCBI under Bioproject PRJNA1117494 and SRA accessions SRR29258991-SRR29258993 and SRR29259000.