PCR bias was previously attributed to intrinsic differences in the amplification efficiency of templates  or to the primer binding energy and kinetics [9, 20]. Our present study, for the first time, revealed the marked bias induced by different polymerase cocktails. It should be note that there were slight differences of Mg2+ and dNTP concentrations between the two cocktails,
but the major factor should be the polymerase. Arezi et al. (2003) found that polymerases showed different efficiencies while amplifying 5 templates varied in length or percentage GC content. The pfu enzyme showed higher efficiency to amplify long templates and high percentage GC content templates. The different efficiently might be related PF-02341066 concentration to the processivity, in addition to the proof-reading function of the enzymes . Although both enzymes used in our present study were high-fidelity enzymes, the PfuUltra VRT752271 cell line II Fusion HS DNA Polymerase was suggested to have enhanced processivity; therefore the two enzymes might have different efficiencies for specific sequences. While amplifying the same 16 S rRNA mixture, we can assume that one enzyme might amplify diverse 16 S rRNA tags at similar efficiency, while the other one might be not, and the determined community structures would be different accordingly.
We can deduce that the community structure at more specific taxonomic levels, e.g. genus or OTU, will change more obviously than the phylum level, as the abundant tags showed so large variances. Nevertheless, we cannot determine which one of the enzymes reflected the real microbial community structure currently, and studies using known 16 S rRNA amalgam as template are warranted. Effect of dilution The present study for the first time explored the effect of template dilution on the microbial Immune system diversity analysis. It is well known
that different soil or sediment DNA extraction methods yield different amount and purity of DNAs . The residual humus and other contaminants in DNA may inhibit the PCR reaction and the DNA is usually diluted for PCR amplification by try and error. Nevertheless, if the dilution affects the diversity analysis has never been explored before. We discussed the template dilution fold rather than the absolute concentration, because 1 gram of different sediment samples might have very different amount of DNA, which should also be considered while analyzing the microbial diversity. Dilution of the template obviously reduced the determined taxa richness, particularly from the 20 fold to 200 fold. The effect of dilution from 1 to 20 fold was less obvious than the above situation, indicating that the 1 fold DNA sample might be saturated and could endure a small fold of dilution. On the other hand, template dilution had few Sotrastaurin ic50 impacts on the microbial community structure determination, as the relative abundance of each unique OTU and the phylum structure showed good similarity among A, B and C groups.