Quality control in multi-tag pyrosequencing of microbial communities

Multi-tag pyrosequencing has become a key method in the analysis of microbial community composition. However, it is well known that kinetic bias during the initial PCR amplification of such microbial communities can dramatically distort amplicon abundance prior to downstream emulsion PCR and pyrosequencing. Here we present a simple protocol combining length-heterogeneity PCR fingerprinting with pyrosequencing to ensure the linearity of microbial community amplification. The method employs a fluorescently labeled reverse primer along with multi-tagged forward primers to initially amplify the microbial community. The resulting labeled amplicons are then fingerprinted, purified, and quantitated prior to emulsion PCR and pyrosequencing. Our data demonstrates: (i) use of this protocol results in a distribution of sequences showing linear amplification following emulsion PCR when compared with the initial length-heterogeneity PCR fingerprints, and (ii) that the added tags and labels do not have a negative effect on overall microbial community profiles.