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 P CR when compared with the initial length-heterogeneity P CR fingerprints, and (ii) that the added tags and labels do not have a negative effect on overall microbial community profiles.