Abstract
Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.
Highlights
The inability to extract nucleic acids (NA) from all populations within a microbial community in sufficient quantity and quality has limited the efficacy of phylogenetic studies in many environments
Initial NA extraction from iron cemented clay using a commercial bead milling process, or liquid nitrogen grinding with a urea based extraction buffer was not productive, and the similarity in color to the refractory East Fork Poplar Creek (EFPC) stream sediments suggested a common source of difficulty
The iron cemented clay had the lowest quantity of acid extractable iron (0.08 mg/g) it contained the highest total iron (38.66 mg/g) following acid digestion, demonstrating that essentially all available iron was in the form of crystalline iron oxides cemented with the clay
Summary
The inability to extract nucleic acids (NA) from all populations within a microbial community in sufficient quantity and quality has limited the efficacy of phylogenetic studies in many environments. Nucleic acid extraction is a well-established source of bias concerning observed microbial communities, most often caused by variation in cell envelope characteristics; eg. A higher DNA yield can indicate low sampling bias and a more complete assessment of the microbial community [4,5]. NA preparation methods differ in NA loss to adsorption, making it difficult to judge microbial community coverage on the basis of NA yield. Because NA adsorption to the environmental matrix makes it impossible to judge extraction bias on the basis of yield, a comparison of procedures should be performed on the basis of the accessed microbial diversity
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