Figure 1

Dual-indexed 16S rRNA gene PCR amplification strategy with heterogeneity spacer primers for sequencing on the MiSeq platform. (A) Dual-indexed PCR amplification primers targeting the V3-V4 hypervariable regions of the 16S rRNA gene contain a heterogeneity spacer region and linker sequence optimized for sequencing on the Illumina MiSeq platform. Using this approach enables sequencing using the standard Illumina HP10 and HP11 sequencing primers allowing for additional sequencing flexibility. (B) Schematic showing the first thirty sequencing cycles of eight mock amplicons prepared using the dual-indexed approach. This diagram illustrates how the index sequence and heterogeneity spacer (colored letters, white background) helps to alleviate the “low sequence diversity” issue associated with the MiSeq platform by creating a more even base composition at each cycle of the run.