Impact of common sample pre-treatments on soil microbial abundance, species, diversity and community composition

Pre-treatments of soil samples prior to DNA-based microbiological community analyses are a common practice but methods vary greatly between laboratories and projects. From sampling to DNA extraction, soils can be either kept fresh, frozen, or dried and it is an open question how these variations affect PCR-based results for quantifying microbial abundance or assessing their diversity by amplicon sequencing. Anticipating that such DNA-based analyses are increasingly demanded for global and long-term environmental monitoring purposes in order to assess the status and potential functionality of soil microbial communities, an evaluation of sampling and pre-treatment procedures is of paramount importance. To elucidated the impact of specific pre-treatment procedures, we collected soils from five different paired-sites (each with cropland and forest) and applied six common pre-treatments: immediate analysis of field-fresh soil samples, and analysis following 14-day pre-incubation of soil samples that were field-fresh, air-dried, oven-dried (40oC), frozen at -20C (gentle freezing), and frozen (direct-freezing: dry ice and subsequently liquid N2). Bacterial, archaeal and fungal abundances were assessed by qPCR of 16S rRNA or ITS1 (for fungi) genes, and diversity as well as microbial community compositions were assessed by amplicon sequencing of the same target DNA using the illumina MiSeq platform.