spyder, a new method for in silico design and assessment of 16S rRNA gene primers for molecular microbial ecology

• Published in: FEMS microbiology letters Vol. 320; no. 2; pp. 152 - 159
• Main Authors: Thomas, Matthew C., Thomas, Dallas K., Selinger, L. Brent, Inglis, G. Douglas
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-07-2011; Wiley-Blackwell; Oxford University Press
• Subjects: 16S rRNA gene; Algorithms; Amino Acid Sequence; bacteria; Bacteriology; Base Sequence; Biological and medical sciences; Communities; Community structure; Computational Biology - methods; Computer-Aided Design; Databases, Nucleic Acid; Deoxyribonucleic acid; Design; Design analysis; Design optimization; DNA; DNA Primers; Ecological monitoring; Environmental Microbiology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Microbiology; Microorganisms; Miscellaneous; Molecular Sequence Data; primer design; Primers; ribosomal database project; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sequence Alignment; Software; universal; bacteria; primer design; universal; 16S rRNA gene; ribosomal database project; Ribosomal DNA; Database; 16S-RNA; Bacteria; Method
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2011.02302.x

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.