Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method

Abstract

BackgroundThe primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform.ResultsThe PfuUltra II Fusion HS DNA Polymerase (Stratagene) with higher fidelity showed lower amount of PCR artifacts and determined lower taxa richness than the Ex Taq (Takara). More importantly, the two polymerases showed different efficiencies for amplifying some of very abundant sequences, and determined significantly different community structures. As expected, the dilution of the DNA template resulted in a reduced estimation of taxa richness, particularly at the 200 fold dilution level, but the community structures were similar for all dilution levels. The 30 cycle group increased the PCR artifacts while comparing to the 25 cycle group, but the determined taxa richness was lower than that of the 25 cycle group. The PCR cycle number did not changed the microbial community structure significantly.ConclusionsThese results highlight the PCR conditions, particularly the polymerase, have significant effect on the analysis of microbial diversity with next generation sequencing methods.

Freshwater bacterioplankton richness in oligotrophic lakes depends on nutrient availability rather than on species–area relationships

“…It should, however, be noted that all three covered the V6 region on the 16S rRNA gene, whereas we targeted the V3 and V4 regions. It has been shown that the choice of primers and other PCR conditions, such as amplicon length, polymerase, annealing temperature, or cycle number affected the estimation of microbial species richness (Liu et al, 2008;Engelbrektson et al, 2010;Wu et al, 2010). Besides, none of the three studies mentioned above applied a method to account for pyrosequencing errors, such as AmpliconNoise (Quince et al, 2011), and, hence, counteracted an overestimation of microbial species richness owing to 454 pyrosequencing (Quince et al, 2009;Reeder and Knight, 2009).…”

Comparison of the Levels of Bacterial Diversity in Freshwater, Intertidal Wetland, and Marine Sediments by Using Millions of Illumina Tags

“…The PCR amplification of bacterial 16S rRNA V6 tags was performed as described in our previous reports (32,34). We used the barcode 967F (CNACGCGAAGAACCTTANC) and 1046R (CGACA GCCATGCANCACCT) primers to amplify the bacterial 16S rRNA V6 fragments.…”

“…In addition, recent meta-analyses showed that there is a higher bacterial and archaeal diversity in inland freshwater than in marine environments (2,3). Nevertheless, PCR conditions, sequencing methods, and 16S rRNA short variable regions being sequenced might affect the meta-analysis, particularly for ␣-diversity, and a reliable comparison should be performed using exactly the same experimental conditions (32,36).…”

The proof of concept that culturomics can be superior to metagenomics to study atypical stool samples

“…We are grateful to the authors for their remarks, and we agree with the biases linked to metagenomics explaining the absence of reproducibility between the different studies about the gut microbiota repertoire and have reported this elsewhere [2][3][4]. Nevertheless, the aim of this study was to compare the most commonly used molecular method to explore the microbiota (pyrosequencing of 16 rRNA amplicons targeting the V6 region) used as a standard with culturomics [5].…”