title: spyder, a new method for in silico design and assessment of 16S rRNA gene primers for molecular microbial ecology.
abstract: Molecular microbial ecology studies are heavily reliant on 'Universal' 16S rRNA gene primers for elucidating microbial community structure and composition, and yet primer design and optimization is often overlooked. Primers that exhibit minor biases due to primer-template mismatches can substantially alter the pool of amplicons from a community DNA sample, resulting in inaccurate conclusions. As a result, it is important that primers are critically evaluated against the most comprehensive data sets available before commencing molecular microbial community studies. We present a user-friendly, multi-platform (e.g. Windows, Linux, Mac) method named spyder for the in silico design and assessment of 16S rRNA gene primers. The method utilizes the Ribosomal Database Project's Probe Match feature coupled with a compact program (available at http://people.uleth.ca/~selibl/Spyder/Spyder.html) that aligns and identifies mismatches between primers and templates. To demonstrate the value of spyder, we assessed commonly used 'Universal' and phyla-specific primers and identified primer modifications that improved the coverage of target organisms by 5-42% as well as removed excessive degeneracies.
authors.affiliation: Agriculture and Agri-Food Canada, Lethbridge, Canada.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Thomas
authors.forename: Dallas K
authors.initials: DK
authors.lastname: Thomas
authors.forename: L Brent
authors.initials: LB
authors.lastname: Selinger
authors.forename: G Douglas
authors.initials: GD
authors.lastname: Inglis
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001483:Base Sequence; D019295:Computational Biology / Q000379:methods*; D017076:Computer-Aided Design; D017931:DNA Primers; D030561:Databases, Nucleic Acid; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D016415:Sequence Alignment; D012984:Software
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S