Soil Sample Size Affects Fungal and Bacterial Community Structure and Richness: New Insights Using High Throughput Sequencing.

In this study we examined the effects of varying soil sample sizes obtained from an Australian agricultural field on bacterial and fungal community composition. DNA extractions from soil sample sizes of 0.5, 1, 5 and 10 g using MoBIO kits and of 10 and 100 g sizes using a proprietary method (SARDI) were used as templates for high-throughput sequencing targeting 16S rRNA gene amplicons on the Illumina MiSeq platform and fungal 28S gene amplicons on the 454 pyrosequencing platform. Sample size was found to significantly affect overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. There were no consistent biases towards any bacterial taxa, though bias was evident in the fungal data. Specifically, smaller MoBIO extractions were biased towards many unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while decreasing replicate variation.