An ideal microbiome profiling method would produce strain level taxonomic classification of organisms and their relative quantities. We demonstrate proof of principle experiments that sequencing methods using amplicons derived from rRNA gene clusters can be designed to differentiate between known strains of Clostridium difficile, a common hospital acquired pathogen. Obtaining consistent and reproducible results depends on overcoming challenges in microbial lysis, amplicon design, PCR primer degeneracy, sample multiplexing technology, and sequence database analysis. Novel approaches developed for each of these challenges are discussed.