Bacterial Mock samples Raw sequence reads. synthetic metagenome

There is much speculation on what hypervariable region gives the highest bacteria specificity in 16s rRNA sequencing. The optimum solution to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene, however, this is not possible with standard library short-read second generation sequencing technology. This analysis examines a new process using six hypervariable regions of the 16S rRNA (V2, V3, V4, V67, V8 and V9) on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were subjected to the 16s Ion Metagenomics Kit ™ (Life Technologies) and a novel analytical pipeline. Two different measures, the Shannon diversity index and the Kullback-Liebler divergence (DKL) were used to infer which region performed most comparably to the projected mock. Three hypervariable regions, V2, V4 and V67 produced the lowest average DKL as compared to the projected mock sample, with the forward and reverse V4 region identifying all 17 family level bacteria. The forward and reverse V9 region gave the highest average DKL identifying only 17% and 53% of the known family level bacteria respectively. The mock samples used in this study allowed for the validation of a new library kit and a novel analytical pipeline while also assessing the performance of multiple different variable regions simultaneously. Future work will include the use of clinical samples from aplastic anemia patients and the presentation of microbial abundance across variable regions at different time points throughout two different courses of treatment.