Taxonomic precision of different hypervariable regions of 16S rRNA gene and annotation methods for functional bacterial groups in biological wastewater treatment

• Published in: PloS one 2013-10, Vol.8 (10), p.e76185
• Main Authors: Guo, Feng, Ju, Feng, Cai, Lin, Zhang, Tong
• Format: Article
• Language: English
• Subjects: Activated sludge; Analysis; Annotations; Bacteria; Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Biodegradation, Environmental; Biological treatment; Biological wastewater treatment; Biotechnology; Blurring; Classification; Consistency; Ecological monitoring; Ecological research; Environmental impact; Environmental monitoring; Foaming; Functional groups; Gene sequencing; Genera; Genes; Genes, rRNA; Genetic Variation; Genomes; Laboratories; Methods; Microorganisms; Molecular Sequence Annotation; Multilocus Sequence Typing; Next-generation sequencing; Phylogeny; Ribonucleic acid; Ribosomal DNA; Ribosomal RNA; RNA; RNA, Ribosomal, 16S - classification; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sequence Analysis, DNA; Sewage - microbiology; Sewage treatment; Sludge; Studies; Taxonomy; Waste treatment equipment; Waste Water - microbiology; Wastewater treatment; Wastewater treatment plants; Water treatment plants
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0076185

High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions - provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.