Nevertheless, as the sequencing accuracy of all next generation sequencing methods decreases at the 3′ end of the reads [19], overlapping of the pair end sequencing reads with 5′ end sequences obviously increases the accuracy of see more the final result. Furthermore, we employed a very stringent pipeline to trim the low quality reads, as we removed all tags with mismatches in the overlapped

region, mismatches with primers, having any N bases, and very short tags. The large number of tags showing mismatches with primers (52,016) had two Anlotinib solubility dmso resources: (i) the impurity of the primers during primer synthesis; and (ii) sequencing error. We suggest that the first one could be the major reason as the quality checking of the primer using mass spectrum showed that there could be nearly 10% of impure primers in the ultra PAGE purified primers (Additional file 3). We found that removing tags with any N bases was very critical, as the 23,222 tags with N bases formed 16,397 unique sequences. Considering that the final number of unique tags was only 67,826, the tags with N bases could contribute a large number of novel unique MLN2238 nmr sequences, but only as singletons or doubletons, therefore to increase the diversity estimation. Although we

may not preclude the sequencing artifacts existing in the final result, we suppose that sequencing error effect has been minimized at the present time and we could explore the PCR effect on the 16 S rRNA deep sequencing methods. Effect of polymerase The polymerase showed significant

effect on both the taxa richness and community structure analysis in our result. Qiu et al. (2001) compared three enzymes with different processitivity and fidelity. They found that the AmpliTaq showed the lowest number PCR artifacts, but not the enzymes with higher fidelity or processitivity. In our study, the two tested polymerases were high fidelity enzymes. The PfuUltra II Fusion HS DNA Etofibrate Polymerase was suggested to have the highest fidelity (20 fold higher than the conventional Taq) and enhanced processitivity (Stratagene manual). The Ex Taq (Takara) had a 4 fold higher fidelity than the conventional Taq. The rarefaction curves of PfuUltra II at the unique distance showed much lower slopes than that of the Ex Taq, indicating that less PCR artifacts were produced using the PfuUltra II enzymes. In addition, while the determined sequences were grouped into 0.03 OTUs, the slopes of rarefaction curves of the two groups showed less pronounced differences, suggesting that a number of the different tags between the two groups could be PCR artifacts, as PCR mutants were suggested to be within 97% similarity with the original sequence [9]. A more important finding of the present study was that the two enzymes showed different community structures, besides different rRNA microbial richness.