Soil, Feces and Skin DNA samples for microbiome characterization using PacBio Long Read Sequencing Raw sequence reads

We describe a simple, flexible and robust workflow for the generation of amplicons for long-read next-generation sequencing platforms. The workflow includes a two-stage PCR protocol that has been previously employed for short-read amplicon sequencing and allows for flexibility in choice of locus-specific primers. In addition, the workflow provides a mechanism for balancing large pools of amplicon libraries without individual reaction cleanups by leveraging small data output short-read sequencing runs. The workflow is demonstrated for sequencing of 16S rRNA gene amplicons generated with 27F-1492R primers from DNA extracted from soil, feces and skin samples on a Pacific Biosciences Revio sequencer.