title: Comparative assessment of next-generation sequencing, denaturing gradient gel electrophoresis, clonal restriction fragment length polymorphism and cloning-sequencing as methods for characterizing commercial microbial consortia.
abstract: Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants.
authors.affiliation: Biological Assessment and Standardization Section, Environment Canada, 335 River Road, Ottawa, Ontario, Canada K1V 1C7. Electronic address: ajith.diassamarajeewa@ec.gc.ca.
authors.forename: A D
authors.initials: AD
authors.lastname: Samarajeewa
authors.affiliation: Biological Assessment and Standardization Section, Environment Canada, 335 River Road, Ottawa, Ontario, Canada K1V 1C7.
authors.forename: A
authors.initials: A
authors.lastname: Hammad
authors.affiliation: National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Quebec, Canada H4P 2R2.
authors.forename: L
authors.initials: L
authors.lastname: Masson
authors.affiliation: Eastern Cereal and Oilseed Research Centre (ECORC), Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, Canada K1A 0C6.
authors.forename: I U H
authors.initials: IU
authors.lastname: Khan
authors.affiliation: Biological Assessment and Standardization Section, Environment Canada, 335 River Road, Ottawa, Ontario, Canada K1V 1C7.
authors.forename: R
authors.initials: R
authors.lastname: Scroggins
authors.affiliation: Biological Assessment and Standardization Section, Environment Canada, 335 River Road, Ottawa, Ontario, Canada K1V 1C7.
authors.forename: L A
authors.initials: LA
authors.lastname: Beaudette
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D019149:Bioreactors / Q000191:economics / Q000382:microbiology*; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D059013:Microbial Consortia; D012150:Polymorphism, Restriction Fragment Length; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D011933:Reagent Kits, Diagnostic
keywords: DGGE; Ion torrent; Microbial consortia; Next-Generation Sequencing; RFLP