Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses

Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental datasets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU dataset derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed the nSSU gene secondary structure was analyzed in several individuals. We observed for the first time that chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in (nSSU) pyro-sequenced environmental datasets. Nonetheless, the region where the chimera forms has to have sufficient conservation to favour hybridization and chimera formation. We anticipate that this study will significantly contribute to a better understanding of the nature and mechanisms involved in chimera formation during the PCR amplification of environmentally derived DNA.