Substitution by inosine at the 3'-ultimate and penultimate positions of 16S rRNA gene universal primers.

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3'-ultimate or the 3'-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities.