Pyrosequencing and the Question of Bacterial Biogeography.

High throughput DNA pyrosequencing of the V9 region of total community of four different soils, three agricultural and one forest, was used to assess overall soil bacterial diversity levels.  The number of bacterial 16S rRNA sequences obtained from each site varied from 26,140 to 53,533 per soil.  Various methods were used to infer that the highest level of bacterial OTU richness, roughly corresponding to species, was 52,000 per gram.  Furthermore, the bacterial diversity of the forest soil appeared to be phylum rich compared to the agricultural soils, which are species rich but phylum poor.  The forest site also showed far less diversity of the archaea with only 0.009% of all sequences from that site being from this group as opposed to 4-12% of the sequences from the three agricultural sites.  To further explore the degree of community composition differences between the soils, sequences were classified and grouped into different phyla, after which the degree of OTU overlap between soils was assessed.  Overall, when OTU’s were defined at 100% sequence similarity,  only 1.5% of OTU’s were found in all four soils, and 87.9% were unique to the soil they were found in. Defined as 97% sequence similarity, only 4.1% of OTU’s were found in all four soils, and 74.4% were unique.  The sensitivity of these conclusions to important variables such as soil storage conditions, primer selection, alignment and classification software, and particularly, study replication, are discussed.