title: Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer.
abstract: Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses.
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Ruegger
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA.
authors.forename: Robin T
authors.initials: RT
authors.lastname: Clark
authors.affiliation: Department of Botany & Plant Sciences, University of California, Riverside, CA 92521, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Weger
authors.affiliation: Department of Pathology and Lab Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Braun
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA. Electronic address: borneman@ucr.edu.
authors.forename: James
authors.initials: J
authors.lastname: Borneman
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D058448:Biota; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D051379:Mice; D010802:Phylogeny; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer
keywords: Bacteria; High throughput sequencing; ITS; Illumina; Internal transcribed spacer; rRNA