Optimization of subsampling, decontamination, and DNA extraction of difficult peat and silt permafrost samples

Abstract

this study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. in addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost researchers to acquire contaminant-free DnA from permafrost with low biomass. Permafrost, i.e. Earth materials below 0 °C for at least two years and up to millions of years, acts as an archive of past environments and ecosystems, preserving biological material as a result of its isolation from atmospheric inputs, low temperatures, and low water activity 1. Ancient DNA derived from long-dead organisms is an important example of such material and has been used for a variety of purposes, ranging from reconstructing human migration patterns to reconstituting the genomes of extinct organisms such as the woolly mammoth and North American horses 2-6. Furthermore, permafrost-dwelling microbes may also play important roles in carbon cycling by conversion of permafrost organic carbon to methane and carbon dioxide, both important greenhouse gasses 7-10. The use of high-throughput sequencing technologies has enriched our understanding of microbial communities in permafrost and ancient DNA. However, these technologies require the extraction of high yields of DNA devoid of contaminants 11. External contamination is particularly problematic in DNA-based approaches due to the high sensitivity in detecting, amplifying and sequencing of DNA. The nature of these contaminants are primarily DNA from humans and background or exogenous DNA of microorganisms 12,13. Obtaining DNA devoid of contaminants from environmental samples, especially from those with low biomass such as permafrost, is often challenging. Such samples are prone to external contamination during drilling and collection in the field and handling in the laboratory, which could lead to misinterpretation of microbial diversity, activity, or ancient DNA studies 14-16. Several methods have been used for permafrost decontamination, such as scraping the outer surface of cores, fracturing of cores followed by clean subsampling from the interior of the core sections (i.e. "disk sampling"), or washing the cores with DNase (e.g. 17...

Climate change, melting cryosphere and frozen pathogens: Should we worry…?

“…The three-step procedure included band saw scrapping, ethanol washing and sterile water washing of the ice cores, before reaching non-contaminated-, “inner ice” layers. The same year, Saidi-Mehrabad et al ( 2020 ) proposed a slightly different procedure to remove contaminants and extract DNA from permafrost samples with low biomass. Again, it combined bleaching, washing, and scraping of the samples, before conducting further experiments.…”

A Case Study for the Recovery of Authentic Microbial Ancient DNA from Soil Samples

“…However, soil or sediment samples are difficult to decontaminate using classical methodologies (e.g., UV irradiation and sodium hypochlorite) routinely applied to bones, hair, or dental calculus [60][61][62]. Removing the outer surface of a soil or sediment core in an aDNA facility is a more suitable decontamination method [14,63,64]. If using coring equipment, there are several contamination tracing approaches [14].…”

RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances

“…To date, a fairly large number of methods for extracting DNA from the soil including commercial kits and open laboratory protocols have been proposed and continue to be developed [1][2][3][4]. Most of the developed methods focused on solving one of the problems, such as high clay or humic substances content.…”

“…1 for these very low amounts (by electrophoresis data) measurements were not taken. 2 The effect is shown only for OMUG, since PCR inhibition is not detected for other soils. * Statistically significant differences in DNA amount between RIAM protocol and the best variant of NucleoSpin Soil kit accordingly to t-test (p < 0.05).…”