Microbial Transcriptome Profiling of Biofilms

Total RNA was isolated from the biofilms using the mirVana miRNA isolation kit (Applied Biosystems). Microbial cells were lysed and RNA was extracted by Acid-Phenol: Chloroform and ethanol precipitation and eluted in nuclease-free water. Ribosomal RNA was depleted and mRNA enriched by modified capture hybridization approach. Enriched mRNA served as a template for the polyadenylation reaction and cDNA synthesis. Microbial libraries were clustered on the Illumina HiSeq platform, and 150 bp paired-end sequencing was performed. The Illumina base-calling pipeline was used to process the raw fluorescence images and call sequences. Raw reads with >10% unknown nucleotides or with >50% low quality nucleotides (quality value < 20) were discarded. Microbial transcripts were quality filtered using SolexaQA++, and aligned against the Human Oral Microbiome Database^{71 (link)} using DIAMOND.^{72 (link)} Aligned sequences were annotated to the KEGG database using Megan 6.^{73 (link)} The metagenomic sequence classifier Kraken^{74 (link)} was used along with our custom tool, kraken-biom, for taxonomic identification. Analysis and visualization of the distribution of operational taxonomic units was performed using QIIME^{75 (link)} and PhyloToAST.^{76 (link)} Bioconductor package for R, DESeq2, was used to perform differential expression analysis of the annotated microbial transcripts.