Efficacy of a 3rd generation high-throughput sequencing platform for analyses of 16S rRNA genes from environmental samples

• PacBio RS and Roche/454 sequencing platforms were compared for 16S rRNA amplicons.
• Based on results from a known isolate, PacBio SMRT chip yielded higher error rates.
• High PacBio error rates inflated richness estimates in environmental samples.
• PacBio, in its current form, does not provide greater phylogenetic resolution.

Longer sequences of the bacterial 16S rRNA gene could provide greater phylogenetic and taxonomic resolutions and advance knowledge of population dynamics within complex natural communities. We assessed the accuracy of a Pacific Biosciences (PacBio) single molecule, real time (SMRT) sequencing based on DNA polymerization, a promising 3rd generation high-throughput technique, and compared this to the 2nd generation Roche 454 pyrosequencing platform. Amplicons of the 16S rRNA gene from a known isolate, Shewanella oneidensis MR1, and environmental samples from two streambed habitats, rocks and sediments, and a riparian zone soil, were analyzed. On the PacBio we analyzed ~ 500 bp amplicons that covered the V1–V3 regions and the full 1500 bp amplicons of the V1–V9 regions. On the Roche 454 we analyzed the ~ 500 bp amplicons. Error rates associated with the isolate were lowest with the Roche 454 method (2%), increased by more than 2-fold for the 500 bp amplicons with the PacBio SMRT chip (4–5%), and by more than 8-fold for the full gene with the PacBio SMRT chip (17–18%). Higher error rates with the PacBio SMRT chip artificially inflated estimates of richness and lowered estimates of coverage for environmental samples. The 3rd generation sequencing technology we evaluated does not provide greater phylogenetic and taxonomic resolutions for studies of microbial ecology.