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 microbiologists to acquire contaminant-free DNA from permafrost with low biomass. IMPORTANCEPermafrost has the capacity to preserve microbial and non-microbial genomic material for millennia; however, major challenges are associated with permafrost samples, including decontamination of samples and acquiring pure DNA. Contamination of samples during coring and post coring handling and processing could affect downstream analyses and interpretations. Despite the use of multiple different decontamination and DNA extraction methods in studies of permafrost, the efficacy of these methods is not well known. We used a biological tracer to test the efficacy of previously published decontamination methods, as well as a bleach-based method we devised, on two chemically and structurally different permafrost core sections. Our method was the only one that removed all detectable tracer. In addition, we tested multiple DNA extraction kits and modified one that is able to acquire pure, PCR amplifiable DNA from silty, and to some extent from peaty, permafrost samples.