Primer modifications for next-generation amplicon sequencing. metagenome

Deep sequencing of 16S rRNA gene amplicons continues to be the most common approach for characterization of complex microbial communities. PCR amplifications of conserved regions of the small subunit ribosomal RNA often employ degenerate pools of primers to enable targeting of a broad spectrum of organisms. One little noticed feature of such degenerate primer sets is the potential for a wide range of theoretical melting temperatures between the primer variants. The melting temperature variation of primers in a degenerate pool could lead to amplification inefficiencies and PCR bias. Thus, we sought to adjust the theoretical melting temperature of each primer individually. Through careful design, we also used this primer modification to introduce nucleotide diversity into next-generation amplicon sequencing protocols without the addition of exogenous templates such as phiX. We demonstrate here the suitability of such primers for microbial community analysis. However, no substantial differences in microbial community structure were revealed when using the shortened primers, though the ideal annealing temperature decreased.