Dataset: dinoRNAV dynamics in the reef-building coral Porites lobata

Sample collection for 'omics analyses (both Symbiodiniaceae LSU and dinoRNAV MCP):
Fifty-four colonies of Porites lobata were tagged on the north shore of Moorea, French Polynesia, spanning nine sites that encompassed three reef zones (fringing, back, and forereef; n = 6 colonies/site, n = 3 sites/reef zone). Each tagged colony was sampled in August 2018 (dry season), March 2019 (wet season), August 2019 (dry), and October 2020 (dry). Samples could not be collected in March 2020 due to the COVID-19 pandemic. Tissue samples were collected for amplicon sequencing; for each P. lobata colony, ~3 to 6 small fragments (1 square centimeter (cm²)) of skeleton/tissue were sampled across the colony surface using bone cutters, placed in a sterile Whirl-Pak® (Nasco), and then preserved in DNA/RNA shield (ZymoResearch, Irvine, California, USA). Samples in the DNA/RNA shield were kept on ice until returning to shore, at which point they were vortexed for 25 minutes at full speed with 5 ceramic beads and 1.35 grams (g) garnet matrix (MP Biomedicals) and then frozen at -40° Celsius (C). These samples were wrapped in aluminum foil and stored at -40°C until further processing. DNA and RNA were extracted from the coral samples, which included mucus, tissue, and skeleton, using enzyme digestions and a ZymoBIOMICs DNA/RNA Kit (ZymoResearch, Irvine, California, USA, following Grupstra et al., 2022).

Symbiodiniaceae LSU:
To identify dominant Symbiodiniaceae lineages, the D1-D2 region of the 28S large subunit (LSU) nuclear ribosomal RNA gene was amplified from coral holobiont DNA. At the Oregon State University Center for Qualitative Life Sciences (CQLS), PCR reactions were conducted using the primers LSU1F_illu and LSU1R_illu with attached Miseq Adapters; cycles were as follows: 95°C for 3 minutes, and then 15 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and finally, 72°C for 4 minutes. PCR clean-up was completed using Agencourt AMPure XP Magnetic Beads. PCR reactions to incorporate Illumina indexing primers were conducted as follows: 95°C for 3 minutes, and then 20 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and finally 72°C for 4 minutes. The resulting PCR product was purified with Agencourt AMPure XP Magnetic Beads, quantified via qPCR using the KAPA library quantification kit (Roche Sequencing Solutions, Pleasanton, CA), pooled in equal molar amounts, and sequenced on the Illumina MiSeq platform with PE300 chemistry.

Amplicons of the LSU gene were processed in RStudio (version 1.1.456) through the DADA2 pipeline (version 1.11.0), which generates unique sequences at single-nucleotide resolution (amplicon sequence variants or ‘ASVs’) (Grupstra et al., 2022). Forward and reverse paired reads were truncated at 210 and 160 base pairs, respectively, based on quality plots. Reads with a total expected error of one or more, or contained Ns, were discarded. ASVs were inferred from unique reads, paired reads were merged, ASVs that did not match a target length of 286 ± 5 were removed, and chimeras were detected and removed. This DADA2 curated table of ASVs were then further collapsed using the LULU package (Frøslev et al., 2017), which merges potentially erroneous ASVs based on sequence similarity and co-occurrence patterns (default parameters of 84% similarity, 90% co-occurrence used here; see https://zenodo.org/record/7552892#.Y_PAa-zMK3I). Taxonomy was assigned to curated ASVs based on BLAST (blastn) searches to NCBI’s nr/nt database, and ASVs that had best matches to non-Symbiodiniaceae were discarded.

dinoRNAV MCP:
The dinoRNAV major capsid protein (mcp) gene was amplified from cDNA (generated from coral holobiont RNA), using a nested PCR protocol with primers from Montalvo-Proaño et al. (2017). Cleaned and normalized libraries were sequenced on the Illumina MiSeq platform using PE300 v3 chemistry at the Oregon State University Center for Qualitative Life Sciences (CQLS). Sequencing and bioinformatic analyses were conducted following Grupstra et al., (2022) using the program vAMPirus (v1.0.1, Veglia et al. 2021). Briefly, after adapter removal, quality filtering, primer removal, read merging, and length filtering, amplicon sequence variants (ASVs) were generated and chimeras removed using VSEARCH with the UNOISE3 algorithm (Rognes et al., 2016; Edgar et al., 2016). Parameters and program information for each of these steps can be found in the vAMPirus config file included as a Supplementary File ("HoweKerr_etal_2023_vAMPirus.config") in Howe-Kerr et al. (2023) and all non-read files used to run the analyses and generate the results can be found at the vAMPirus Zenodo (https://doi.org/10.5281/zenodo.7552892). To collapse some of the diversity associated with the high mutation rate of ssRNA viruses, ASVs were then translated and aligned into unique amino acid types ('aminotypes') using VirtualRibosome (v2.0, Wernersson et al., 2006) and CD-HIT (v.4.8.1, Fu et al., 2012).

Temperature:
Water temperatures were measured every two hours year-round at each site for the duration of the study using a HOBO® temperature logger.

Colony images:
Photographs of each colony were taken at each sampling point and used in visual assessments to determine if a colony remained apparently healthy or experienced partial mortality over the course of the study; a third category ('ambiguous') was used to describe colonies for which health trajectory could not be determined based on the images available. Colonies were considered to have experienced partial mortality if there was a clear loss of live tissue surface area between August 2018 and October 2020; colonies were considered to have remained apparently healthy if there was an increase in live tissue in images taken over the course of the assessed period or if there was no clear change in live tissue surface area. Final health determination was based on assessments by three separate observers, who assessed the images separately and blindly without knowing what reef zone or site a set of images was collected from. Differences in coral health among reef types and sites were assessed with Chi-squared tests.

Known issues or problems:
Due to logistical issues, HOBO® temperature logger data are not available for October 2020.