Ultra-high throughput multiplexing and sequencing of long amplicon regions on the Illumina HiSeq 2500 platform

Amplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and projects have grown in size and scope, greater sample multiplexing is becoming necessary while maintaining high quality sequencing. Here, modifications to the Illumina HiSeq 2500 platform are described that afford 300 bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To make this method feasible and flexible to different amplicon regions, a 2-Step PCR amplification protocol is also described that allows for amplification and sequencing of different amplicon regions, and that improves amplification success from low bacterial bioburden samples.