2.3. Transcriptome-based gene expression analysis

RNA-seq data generated from the sampled different development stages (PRJNA808620), different tissues (PRJNA664867), and clams with different resistance after V. anguillarum challenge (PRJNA811359) were used to examine the expression profiles of TLR genes in R. philippinarum [28,32,38]. Eight tissues including adductor muscle, mantle, foot, gill, pipe, digestive gland, gonad, and labial palp were collected from 3 individuals [32]. RNA-seq data of the different developmental stages including fertilized egg (FE), 1st polar body (PB1), 2st polar body (PB2), 2-cell (TC), 8-cell (EC), blastula (B), gastrula (G), trochophora (T), D shape larva (D), umbo veliger (U), pediveliger (P), single pipe juvenile (S), and juvenile(J) [39]. The expression characteristics of the TLR genes were normalized and represented in the form of RPKM (Reads per Kilobase of exon model per Million mapped reads). The P-values were adjusted using the Benjamini and Hochberg method [30]. Differences were considered significant at P< 0.05. To render the data suitable for cluster displays, absolute Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) values were divided by the mean of all of the values, and the ratios were transformed by log2 (ratio) [30]. A heat map was generated using R-4.0.2 software (https://www.rproject.org/) with the fold-change values [30, 40].