Preservation of nucleic acids by freeze-drying for next generation sequencing analyses of soil microbial communities

Aims Soil sample preservation is a challenging aspect in molecular studies on soil microbial communities. The demands for specialized sample storage equipment, chemicals and standardized protocols for nucleic acid extraction often require sample processing in a home laboratory that can be continents apart from sampling sites. Standard sampling procedures, especially when dealing with RNA, comprise immediate snap freezing of soils in liquid nitrogen and storage at - 80 degrees C until further processing. For these instances, organizing a reliable cooling chain to transport hundreds of soil samples between continents is very costly, if possible at all. In this study we tested the effect of soil sample preservation by freeze-drying with subsequent short-term storage at 4 degrees C or ambient temperatures compared to -80 degrees C freezing by comparative barcoding analyses of soil microbial communities. Methods Two grassland soil samples were collected in Central Germany in the Biodiversity Exploratory Hainich-Dun. Samples were freeze-dried or stored at -80 degrees C as controls. Freeze-dried samples were stored at 4 degrees C or ambient temperature. Investigated storage times for both storage temperatures were 1 and 7 days. Total DNA and RNA were extracted and bacterial and arbuscular mycorrhizal (AM) fungal communities were analyzed by amplicon 454 pyrosequencing of the 16S (V4-V5 variable region) and 18S (NS31-AM1 fragment) of ribosomal RNA (rRNA) marker genes, respectively. Important Findings Bacterial communities were sufficiently well preserved at the rDNA and rRNA level although storage effects showed as slightly decreased alpha diversity indices for the prolonged storage of freeze-dried samples for 7 days. AM fungal communities could be studied without significant changes at the rDNA and rRNA level. Our results suggest that proper sampling design followed by immediate freeze-drying of soil samples enables short-term transportation of soil samples across continents.