Bioinformatics and statistics analysis
Raw RNA-Seq data were quality checked using FastQC v0.11.8 and then pre-processed using Cutadapt v2.8 to remove adapter sequences and poor-quality bases. The pre-processed sequencing reads were processed using QIIME2 v2019.10.0 and were denoised using DADA2 and converted to amplicon sequence variants (ASVs). Non-bacteria taxa e.g., eukaryota, chloroplast and mitochondria in the sequence output by DADA2 were removed. Taxonomic assignments were made using the Silva v132 annotation. DADA2 detected 2456 ASVs of which 278 bacterial taxa were recovered. A total of 2653288 feature counts were detected from all samples with an average of 23072 count per sample (range: 2101 - 318474 counts). A filter was applied to remove counts < 2 and minimum prevalence of 10% per sample with low variance (5%) (