Transcriptome Sequencing of Microbial Organism from Clinical Samples. mixed culture

The goal was to establish a robust and scalable RNA-seq process applicable to cultured bacteria as well as to complex community transcriptomes. To provide a benchmark for method evaluation, we prepared RNA from three well characterized organisms (Prochlorococcus marinus, Escherichia coli, and Rhodobacter sphaeroides) that cover a wide range of base compositions (30%%, 51%%, and 69%% genome GC content, respectively). We prepared total RNA from each organism and used these samples separately or as a “PER” pool (mixed 1:1:1 by mass) of input material. The PER pool was used to 1) evaluate different rRNA depletion methods, 2) accurately maintain relative representation of transcript sequences, 3) work well with RNA of varying quality, and 4) be highly reproducible. To this end, we evaluated rRNA depletion methods and chose a protocol that eliminates rRNA reads efficiently and robustly, largely irrespective of the quality of the RNA input sample, and the method was then applied to clinical samples