Commercial DNA extraction kits impact observed microbial community composition in permafrost samples

Abstract

The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real‐time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA® SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil® (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta‐G‐Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed β‐ and γ‐Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead‐beating lysis extraction kits.

Impact of Different Methods of Soil Sampling and DNA Extraction on the Identification of Soil Bacterial Abundance under Elevated CO2

“…The greater amount of DNA extracted by the Fast kit is consistent with much higher 16s rRNA gene copy numbers using this kit. Similar to our studyVishnivetskaya et al (2014) [38] extracted more DNA using the Fast kit than the MoBio kit from permafrost soils in the Arctic; although in their study both the Fast and MoBio kits extracted high quality DNA (see Table 1 of Vishnivetskaya et al (2014) for comparison of the kits) [38]. The DNA extracted by the MoBio kit in our study was of a higher quality than that extracted using the Fast kit.…”

Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

“…Even if an equimolar mix of samples was used for sequencing, significantly different numbers of reads were obtained depending on the primer set used (see Table S4), as previously noted in other projects (A. Dheilly, Biogenouest Platform, Rennes, France, personal communication). This variability may reflect differences in the sample titration in the equimolar mix (16) or may have been due to the fusion primer set used. Indeed, association of primers with tag sequences and pyrosequencing adaptors may reduce the hybridization efficiency on DNA capture beads, which is required for emulsion PCR and sequencing, and/or may be a disadvantage in amplification.…”

“…While molecular analyses provide a less biased picture of environmental microbial communities than culturebased methods, each step of the analysis needs to be taken into account. Indeed, the DNA extraction method, PCR amplification, or data analysis can all lead to distortions of the compositions of analyzed samples (10)(11)(12)(13)(14)(15)(16)(17)(18).…”

Effects of simulated spring thaw of permafrost from mineral cryosol on CO2 emissions and atmospheric CH4 uptake

“…Enumeration of the bacterial abundance in these permafrost samples by fluorescence in situ hybridization yielded 9.7 × 10 8 cells g À1 [Vishnivetskaya et al, 2014]. The assumed growth yield represents a maximum increase of at most 1 to 4% of the observed population since much of the anabolic activity during the initial thaw probably goes to cellular repair [Price and Sowers, 2004].…”