This post contains the bioinformatic pipeline used for analyzing Stylophora pistillata mRNA sequences, to reveal gene expression dynamics across shallow (5 m) and mesophotic (45 m) reefs under ambient (8.2 pH), intermediate-low (7.8 pH), and low (7.6 pH) pH conditions. All the scripts employed for the analysis can be found in the project electronic notebook.

1) RNA-Seq reads quality filtering and mapping

Total RNA was extracted from shallow and mesophotic S. pistillata fragments collected in Eilat (Red Sea). RNA-seq libraries were prepared using an in-house protocol at the Weizmann Institute of Science (Israel).

Raw sequence data - (NCBI SRA) - Raw Illumina sequence data (fastq format) for the 6 coral colonies succesfully sequenced in this study (accession code PRJNA701170).

Quality filtering and mapping - Details the processing and analyses of the S. pistillata transcriptome sequencing data. RNA-Seq reads processing included adapter trimming using Cutadapt v1.15 (Martin, 2011) and quality filtering using Trimmomatic v0.3 (Bolger et al., 2014). Reads were aligned to the host genome assembly NCBI GCA_002571385.1 using STAR v2.5 (Dobin et al., 2013; Voolstra et al., 2017).

2) Species identification

Coral host and symbiont species identification - High quality reads were blasted using Diamond v2.0.7 (Buchfink et al., 2015) against the NCBI and Reefgenomics genome-based proteomes databases of Symbiodiniaceae species Symbiodinium microadraticum, Cladocopium goreaui, and Fugacium kawagutii (formerly Symbiodinium spp. clades A, C1, and F, respectively (LaJeunesse et al., 2018)), as well as to the databases of Cnidaria, selected stramenopiles/alveolates/Rhizaria and Metazoa.

3) Differential expression

Coral host differential expression - DE analysis was conducted using Bioconductor DEseq2 v1.30.1 (Love et al., 2014) by a) analyzing mesophotic and shallow samples separately, considering a single factor (pH) and three levels (8.2, 7.8, 7.6), and b) analyzing samples from each pH separately, considering a single factor (depth) and two levels (shallow, mesophotic).

4) Functional enrichment

Coral host functional enrichment - Genes biological terms were assigned based on S. pistillata Uniprot, KEGG and Trinotate annotations (Bryant et al., 2017). Enrichment analysis was conducted in Bioconductor GOSeq v1.42.0, as described previously (Malik et al., 2020).

5) SNPs characterization

Coral host SNPs characterization - SNPs analysis was conducted using the recommended RNA-Seq SNPs practice of the Broad Institute. Alignment biases were removed from the STAR-aligned reads, variant calling was performed using the GATK HaplotypeCaller v3.5 (McKenna et al., 2010), and finally variant filtration and annotation was carried out following (DePristo et al., 2011). Genetic differentiation among depths was assessed using Identity By State (IBS) analysis, which was conducted using SNPRelate package v1.24.0 in R. The degree of genetic differentiation was also determined by estimating the fixation index (Fst)(Weir & Cockerham, 1984) using the HIERFSTAT package v0.5-7 in R, between samples located at the same depth and between samples at different depths.