title: High-resolution phylogenetic microbial community profiling.
abstract: Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Esther
authors: E
authors: Singer
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Brian
authors: B
authors: Bushnell
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.|USDA-ARS, Albany, CA, USA.
authors: Devin
authors: D
authors: Coleman-Derr
authors: Pacific Biosciences, Menlo Park, CA, USA.
authors: Brett
authors: B
authors: Bowman
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Robert M
authors: RM
authors: Bowers
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Asaf
authors: A
authors: Levy
authors: University of British Columbia, Vancouver, BC, Canada.
authors: Esther A
authors: EA
authors: Gies
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Jan-Fang
authors: JF
authors: Cheng
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Alex
authors: A
authors: Copeland
authors: Newcastle University, School of Biology, Newcastle upon Tyne, UK.
authors: Hans-Peter
authors: HP
authors: Klenk
authors: University of British Columbia, Vancouver, BC, Canada.
authors: Steven J
authors: SJ
authors: Hallam
authors: Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors: Philip
authors: P
authors: Hugenholtz
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Susannah G
authors: SG
authors: Tringe
authors: US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.
authors: Tanja
authors: T
authors: Woyke
mesh_terms: D001105:Archaea / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001955:British Columbia; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D060106:Lakes / Q000382:microbiology; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D012336:RNA, Ribosomal, 16S