An Improved Dual-Indexing Approach for Multiplexed 16S Microbial Sequencing on the Illumina MiSeq Platform

The ability to take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of novel methods to overcome technical limitations inherent to these platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina Miseq platform and often generates low quality sequences. Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. This approach addresses the known “low sequence diversity” issue associated with sequencing 16S rRNA gene amplicons on this platform and provides a flexible and cost-effective option.