Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

Abstract

dModern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actinobacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms. Soil is one of the most complex and diverse microbial habitats, with 1 g containing up to 10 10 cells and 10 4 bacterial species (1, 2). While DNA sequences provide information about the total microbial community, RNA can be used to analyze the fraction of microorganisms that has the capacity to actively synthesize proteins (3-5). The ratio of bacterial 16S rRNA transcripts to 16S rRNA gene copies has been used as an indicator of the potential specific activity since it reflects the cellular ribosome content (6, 7). However, nucleic acid extraction from soils is affected by insufficient cell lysis or losses during extraction, variable reproducibility, and various effects of soil pH, clay content, and organic carbon content (8-13), and coextracted organic compounds can inhibit DNA polymerase and reverse transcriptase employed in downstream molecular analyses (14).Direct nucleic acid extraction methods are based on the lysis of cells inside the soil matrix, whereas indirect methods commence with the isolation of bacterial cells from soil prior to nucleic acid extraction (8). Indirect methods typically yield longer nucleic acid fragments which are useful for metagenomic studies (10, 15) but often result in a significantly lower yield and diversity of nucleic acid molecules (10, 16). Existing protocols for the direct extraction of DNA and RNA from soils (13, 17-25) have partly been evaluated (19,21,24,(26)(27)(28)(29)(30)), but only a few studies have compared extraction efficiencies of methods recovering both DNA and RNA (21, 24). In particular, these methods have so far not been compared with extraction protocols optimized for either DNA or RNA alone. Information about the effects of different extraction methods on the phylogenetic analysis is scant (31, 32). As a result, ...

Effect of the extraction and purification of soilDNAand pooling ofPCRamplification products on the description of bacterial and archaeal communities

“…As mentioned above, Wüst et al . () observed that extraction methods which employed phenol–chloroform–isoamyl alcohol favoured the lysis of some phyla, specifically Alphaproteobacteria, Actinobacteria and Chloroflexi, which are different than the taxa which were favoured in present study by G‐protocol that also use these solvents. Another study (Yuan et al .…”

“…With respect to the inclusion of phenol–chloroform–isoamyl alcohol, Wüst et al . () observed that all four methods (of eight methods compared) which employed these solvents recovered higher percentages of similar taxas in the DNA extracts. They also suggested that CTAB‐based methods are less efficient in DNA extraction than methods that use SDS.…”

“…; Wüst et al . ). For example, the Meta‐G‐Nome TM DNA Isolation Kit, which uses an indirect extraction method with no bead beating, was the most distinct from three other kits that use direct bead beating cell lysis (Vishnivetskaya et al .…”

“…Interestingly, the comparison of eight DNA extraction protocols, all using direct bead beating lysis, showed that the observed microbial communities with commercial kits tended to group together in comparison with lab‐customized protocols (Wüst et al . ). Thus, it is also possible that we used two contrasting methods while other studies (Terrat et al .…”

“…Wüst et al . () concluded that ‘single standardized protocol for nucleic acid extraction that would be equally efficient for different soil types, target organisms, and nucleic acid types does not exist’. While this conclusion might be true, it is also possible that more effort is needed to achieve this goal.…”