CGIG_SALINITY_RNASEQ_PRJNA756710

Primary-analysis sample count: 12. The counted unit and independent experimental units are described under Biological replication; counts may represent animals, pools or libraries.

Treatment

Intramuscular injection of Vibrio alginolyticus (50 uL of 5 x 10^8 CFU/mL) followed by transfer to 10 ppt seawater (“10‰Vibrio”)

Control

The same Vibrio alginolyticus injection followed by holding at the 30 ppt acclimation salinity (“30‰Vibrio”)

Biological replication

12 RNA-seq libraries used for the contrast: 6 at 30 ppt and 6 at 10 ppt, three per salinity at each of 12 h and 48 h. Each library pools equal gill tissue from three oysters taken from one tank. Two further 0 h libraries (SRR15559848, SRR15559847) are shared pre-transfer baselines with no salinity contrast and are not used.

Phenotype interpretation

Higher mortality after Vibrio infection is unfavorable; infected oysters reached 100% mortality at 10 ppt versus 33% at 30 ppt over 12 days (log-rank p < 0.01, n = 30 per tank in triplicate). AREE records the gill transcriptional response to low salinity during infection, not survival per library.

Analysis status

raw_reanalysis_harmonized

Quality control and results

All 260,215,874 read pairs quantified with Salmon 2.3.4 against GCF_963853765.1/RS_2024_06; mapping rates 70.78-80.24% (median 76.97%), consistent with the 73.9-82.5% the publication reports against GCA_902806645.1 and flat across arms (30 ppt 77.7% versus 10 ppt 76.6%, Welch p = 0.49); 3,045 of 21,098 tested genes reach FDR < 0.05 (1,825 low-salinity-higher, 1,220 lower); PC1 explains 27.9% of variance and separates all six 10 ppt libraries from all six 30 ppt libraries; low_salinity_h12_2 is displaced on PC2 and has the most genes above the Cook’s-distance threshold (84), but leaving it out gives effects correlated 0.929 with the primary model, preserves the direction of all 3,045 primary significant genes and retains 2,618 of them at FDR < 0.05

Limitations

  • Every sequenced oyster was infected with Vibrio alginolyticus, so the contrast estimates the effect of low salinity during infection, not of low salinity alone. The uninfected, sham-injected controls held at 30 ppt were used for the survival curve only and were not sequenced. stressor_class is salinity because salinity is the only factor that differs between the contrasted arms.
  • The primary AREE contrast pools 12 h and 48 h and blocks on sampling time (~ timepoint + condition). It estimates a low-salinity effect averaged over the two times, whereas the publication tested each time separately; each time-by-salinity cell has only three libraries.
  • Each salinity arm used three tanks, and a pool of three oysters was taken from each tank at each time, so the 12 h and 48 h libraries with the same replicate number may share a tank. The public metadata do not state that replicate numbers correspond to tanks, so the model does not pair them.
  • Oyster age or size class is not given beyond an average wet weight of 18.2 +/- 2.8 g, so life_stage is not recorded. Temperature during the challenge is not reported.
  • The library names use C23 for the normal-salinity arm, but the publication and BioSample titles give the normal salinity as 30 ppt; C10 matches the 10 ppt arm.
  • Condition, time and replicate come from the NCBI BioSample titles. They agree with the library names (C10/C23 followed by 1-3 for 12 h and 4-6 for 48 h), and the raw read count of every library matches publication Supplementary Table 3.
  • The publication aligned reads with HISAT2 to GCA_902806645.1, counted them with featureCounts and ran DESeq2 per time point (padj < 0.05, |fold change| > 1). It reports 3,038 DEGs at 12 h (1,731 higher, 1,307 lower at 10 ppt) and 3,187 at 48 h (1,656 higher, 1,531 lower) in Supplementary Table 4, keyed by RefSeq LOC identifiers. Only significant genes are listed, so the table cannot be used as a full ranked result.
  • The companion 16S rRNA amplicon data for the digestive gland are in the separate BioProject PRJNA756403 and are outside this record.
  • Against the publication’s per-time DEGs (Supplementary Table 4), 5,206 of 6,225 LOC identifiers map directly to a current GeneID. Of those tested here, 96.0% (12 h) and 95.7% (48 h) change in the same direction in the pooled AREE model, fold changes correlate at r = 0.784 and 0.759, and none is significant in the opposite direction; 44% reach FDR < 0.05 in the pooled model.
  • rRNA genes are over-represented among low-salinity-higher genes (10 observed versus 0.8 expected; adjusted p = 1.2e-10), which points to a difference in residual rRNA between arms rather than a biological response; those loci should not be interpreted.

Sources