Bias in Bacterial Diversity as a Result of Nycodenz Extraction from Bulk Soil

Nycodenz density centrifugation allows extraction of both culturable and unculturable bacterial cells from soil, which can be used for further analysis; however there is a lack of information concerning the efficiency of this method. The aim of this study was to investigate the efficiency of Nycodenz extraction from soil. Bacterial cells were extracted from three soil plots from the Danish CRUCIAL field trial using a Nycodenz protocol. DNA was extracted from soil, from Nycodenz extracted cells, and from the soil pellet left after Nycodenz extraction. The 16S rRNA gene was amplified from the extracted DNA. The PCR amplicons were tagged sequenced using a GS FLX pyrosequencing system. Sequences were processed and analyzed by using the Ribosomal Database Project’s (RDP) Pyrosequencing Pipline tools. In this study, we show that extraction of bacteria from soil using Nycodenz density centrifugation is biased and results in over- or underrepresentation of common bacterial phyla. Rarefaction curves, analysis of similarity, multidimensional scaling plots and analysis of variance showed that the diversity in the Nycodenz extracted sample was lower than in the original soil or soil pellet. In addition, RDP classifier results showed a clear over- or underrepresentation of certain phyla after Nycodenz extraction. A mathematical model was constructed to estimate how many sequences would be necessary to find 95% of all operational taxonomic units (OTUs) in the soil. The model shows that the soil contains approximately 29 400 OTUs and that just 351 500 sequences are needed to cover 95% of the bacterial biodiversity. The equivalent of one full standard GS FLX run.