The fields of biology and environmental health are undergoing an unprecedented transformation in the way communities are characterized and studied. One of the catalysts for this change has been the advent of high-throughput sequencing (aka next-generation sequencing). Though high-throughput sequencing is a relatively new concept, the principles behind it are fairly straightforward (PCR and pyrosequencing) and have been in the published literature since the early 1990s. Herein, I will highlight some of the more commonly encountered issues involving experimental design as well as important considerations for metagenomic sample collection (both quantity and quality) and data handling for high-throughput sequencing using the Roche/454 Life Sciences genome sequencing platform.