High-resolution single-molecule long-fragment rRNA gene amplicon sequencing for uncultured bacterial and fungal communities

Although several large-scale environmental microbial projects have been initiated in the past two decades, understanding of the role of complex microbiotas is still constrained by problems of detecting and identifying unknown microorganisms1-6. Currently, hypervariable regions of rRNA genes as well as internal transcribed spacer regions are broadly used to identify bacteria and fungi within complex communities7, 8, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length9-11. Direct sequencing of full length rRNA genes is currently limited by read length using second generation sequencing or sacrificed quality and throughput by using single molecule sequencing. We developed a novel method to sequence and assemble nearly full length rRNA genes using second generation sequencing. Benchmarking was performed on mock bacterial and fungal communities as well as two forest soil samples. The majority of rRNA gene sequences of all species in the mock community samples were successfully recovered with identities above 99.5% compared to the reference sequences. For soil samples we obtained exquisite coverage with identification of a large number of putative new species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.