Metagenome complexity and template length are the main causes of bias in PCR-based bacteria community analysis.

Multitemplate PCR is used widely for the study of microbial community diversity. Although such studies have established the abundance of different groups within many natural ecosystems, these reports are limited by uncertainties such as bias and artifacts in the PCR. Bias which is introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. In this study, factors leading to the bias of the multitemplate PCR in bacterial communities were examined and optimized. Comparisons between PCR cycle parameters, DNA polymerases, PCR primer degeneracy, and 16S rRNA gene fragments GC content, revealed that annealing temperatures and DNA structure are predominant factors contributing to the observed bias. Pre-digestion of metagenomic DNA with the restriction enzyme Sau3A I and decreased annealing temperature reduced the bias significantly. The application of these optimized conditions to the ten-species model community in a soil sample verified the validity of these treatments.