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FEMS Microbiol Lett. 2011 Jul;320(2):152-9. doi: 10.1111/j.1574-6968.2011.02302.x. Epub 2011 May 25.

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

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Agriculture and Agri-Food Canada, Lethbridge, Canada.


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 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.

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