Analyzing the oral microbiome: Does utilization of multiple 16S rRNA regions allow for additional bacterial identification?

Authors

• A Ranucci
• H Kim
• J Barb
• A Oler
• N Chalmers
• A Cashion
• G Wallen
• N Ames

Abstract

The oral microbiome is a complex microbiota with a variety of implications for understanding disease and infection. Accurate and sensitive sequencing techniques are integral to the effective investigation of the microbial community in a clinical setting. With the advent of Next Gen Sequencing, it is possible to identify bacteria by sequencing different hypervariable regions within the 16S rRNA gene, however, questions arise as to which variable region is the most effective for identifying oral bacteria. This study compares two different methods using the Ion Torrent Personal Genome Machine (Life Technologies.) One approach uses primers specific to the V4 region while the other utilized the 16s Ion Metagenomics Kit ™ (Life Technologies) which contains primers for the V2, V3, V4, V67, V8 and V9 regions. The purpose of this analysis was to determine the sensitivity of each region and to ascertain whether or not examining multiple hypervariable regions would yield more information than V4 alone. Tongue brushings from an aplastic anemia patient undergoing stem cell transplantation were obtained and subjected to the two aforementioned methods. Raw reads were run through a pipeline using tools from Mothur, UPARSE, and QIIME. Preliminary data shows the 6 region kit allowing for the identification of more operational taxonomic units than V4 alone, specifically down to the species level. This work has allowed us to explore the efficacy of the multi-region approach and justified the use of this sequencing technique for future work in order to get the highest specificity and most bacteria identified.

Scientific Focus Area: Computational Biology

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