Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments
Despite more than three decades of progress, extraction of high molecular weight (HMW) DNA from soils with high clay or iron oxide cemented clay content has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage in DNA libraries. Several nucleic acid extraction procedures that included liquid N2 grinding and bead milling were compared for preparation of HMW DNA from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions (eg. Na2SO4 and NH4H2PO4) 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 and low in humus. 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™ soil DNA system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots based on 16S rRNA (V1/V3 region) pyrosequencing libraries from A-horizon and clay soils showed an ~80% and ~400% larger accessed diversity compared to a previous grinding protocol or 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. Additionally, some OTU’s having more than 100 sequences in libraries prepared from DNA acquired using the PB desorption method were absent in samples extracted using the PowerLyzer™ reagents or prior lysis methods.