CGIG_HEAT_RNASEQ_PRJNA593309

Primary-analysis sample count: 5. The counted unit and independent experimental units are described under Biological replication; counts may represent animals, pools or libraries.

Treatment

Acclimated 10 days at 29 C (ramped 2 C per day from 21 C) and sampled 12 h after the start of OsHV-1 muVar cohabitation

Control

Acclimated at 21 C and sampled 12 h after the start of OsHV-1 muVar cohabitation

Biological replication

5 oysters from 6 libraries: three at 21 C, each from a different tank, and two at 29 C, because two of the three 29 C libraries are the same oyster and were summed. The publication describes one oyster per tank, so the duplicated 29 C oyster means either one tank was sampled twice or one RNA was sequenced twice.

Phenotype interpretation

Survival 14 days after cohabitation was higher at 29 C (85.7%) than at 21 C (52.4%), and viral load rose more slowly at 29 C. AREE records the whole-oyster transcriptional difference at 12 h, not survival per library.

Analysis status

raw_reanalysis_harmonized

Quality control and results

All 12 downloaded libraries quantified with Salmon 2.3.4 against GCF_963853765.1/RS_2024_06; mapping is lower at 29 C (44.5-47.6%) than at 21 C (53.5-57.8%) at 12 h. Expressed-SNP allele frequencies at 801 sites show oshv_29C_12h_1 and oshv_29C_12h_2 are one oyster (mean difference 0.025 against 0.029 expected for one RNA resequenced; every other pair differs by 0.28 or more), so their libraries were summed and the contrast is 3 oysters at 21 C against 2 at 29 C. ~ condition: 198 of 14,943 tested genes reach FDR < 0.05 (30 higher, 168 lower at 29 C), against 684 when the duplicated oyster is counted twice. PC1 explains 49.3% of variance. Lower at 29 C are cholesterol transport (NPC intracellular cholesterol transporter) and other metabolic genes, in line with the publication’s down-regulation of metabolism at 29 C; its up-regulated E3 ubiquitin ligase and synaptic receptor genes are not significant here. The publication reports 1,189 genes at 12 h with three libraries per temperature.

Limitations

  • Both arms are infected, so the contrast is temperature in the context of early OsHV-1 infection, not temperature alone; before infection the publication finds only nine genes differing between 29 and 21 C.
  • One whole oyster per tank was sequenced at each time point, with three replicate tanks per temperature (45 l tanks), so libraries are tank-level replicates and the contrast has three tanks per arm.
  • The two AREE records from this study use different oysters but the same three 21 C tanks (the 21 C 12 h oysters of CGIG_HEAT_RNASEQ_PRJNA593309 and the 21 C 0 h and 48 h oysters of CGIG_OSHV1_RNASEQ_PRJNA593309), so both carry the dependence group PRJNA593309_21C_tanks and count as one replication group.
  • The BioSamples give a collection date of 2019-05-19, but the publication dates the experiment to May 2016; the experimental groups follow the sample aliases (Cg_<CTR | OsHV>hpc_), which agree with the publication’s design.
  • The study also deposited libraries at 26 C and at 24 and 96 h; the publication does not analyse the 26 C libraries, and AREE uses only the time points of its two contrasts.
  • The publication aligned reads with STAR to the Ensembl oyster genome v9.38 and tested with edgeR; AREE quantifies against GCF_963853765.1/RS_2024_06 with Salmon and DESeq2.
  • Two of the three 29 C libraries at 12 h are the same oyster by expressed-SNP genotype, so this record has two independent 29 C oysters, little power, and fewer significant genes than the publication, whose three-replicate analysis counted that oyster twice.
  • Mapping is about eight points lower at 29 C than at 21 C at 12 h, a systematic shift that may reflect more viral or unspliced RNA at 29 C or a batch difference.

Sources