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

Abstract

25This study aims to act as a methodological guide for contamination monitoring, 26 decontamination, and DNA extraction for peaty and silty permafrost samples with low 27 biomass or difficult to extract DNA. We applied a biological tracer, either only in the 28 field or both in the field and in the lab, via either spraying or painting. Spraying in the 29 field followed by painting in the lab resulted in a uniform layer of the tracer on the core 30 sections. A combination of bleaching, washing, and scraping resulted in complete 31 removal of the tracer leaving sufficient material for DNA extraction, while other widely 32 used decontamination methods did not remove all detectable tracer. In addition, of four 33 widely used commercially available DNA extraction kits, only a modified 34ZymoBIOMICS TM DNA Microprep kit was able to acquire PCR amplifiable DNA. 35Permafrost chemical parameters, age, and soil texture did not have an effect on 36 decontamination efficacy; however, the permafrost type did influence DNA extraction. 37Based on these findings, we developed recommendations for permafrost microbiologists 38 to acquire contaminant-free DNA from permafrost with low biomass. 39 IMPORTANCE: 40Permafrost has the capacity to preserve microbial and non-microbial genomic material for 41 millennia; however, major challenges are associated with permafrost samples, including 42 decontamination of samples and acquiring pure DNA. Contamination of samples during 43 coring and post coring handling and processing could affect downstream analyses and 44 interpretations. Despite the use of multiple different decontamination and DNA extraction 45 methods in studies of permafrost, the efficacy of these methods is not well known. We 46 used a biological tracer to test the efficacy of previously published decontamination 47 methods, as well as a bleach-based method we devised, on two chemically and 48 structurally different permafrost core sections. Our method was the only one that 49 removed all detectable tracer. In addition, we tested multiple DNA extraction kits and 50 modified one that is able to acquire pure, PCR amplifiable DNA from silty, and to some 51 extent from peaty, permafrost samples. 52 53 54 55

Permafrost Microbial Community Structure Changes Across the Pleistocene-Holocene Boundary

“…These three selected intervals were vertically cut into 1/3 and 2/3 subsections with the aid of a masonry saw. The 2/3 sections were used for DNA extraction, while the 1/3 sections were used for chemical analyses (Saidi-Mehrabad et al, 2020).…”

“…Selected intervals of DHL_16_1, DHL_16_2, and DHL_16_3 were sub-sectioned for soil chemical analysis based on a previously described method (Saidi-Mehrabad et al, 2020). Briefly, the 1/3 core sections were cut into 1 cm 3 cubes with a hand saw in a 4 • C cold room for measuring the basic chemical parameters of these samples, including organic carbon (OC), pH and gravimetric water content (GW) (Saidi-Mehrabad et al, 2020). Total carbon (TC) and total nitrogen (TN) were measured from 0.5 g of subsections based on a dry combustion method with the aid of an Elemental Analyzer (EA 4010; Costech International Strumatzione, Italy) (Sparks et al, 1996).…”

“…Prior to DNA extraction the 2/3 sections associated with the DHL_16_1, DHL_16_2, and DHL_16_3 intervals were cut into ∼6 × 3.5 × 2.5 cm rectangular pieces (n = 16) and the exterior of these rectangles were painted with a total of 3 × 10 8 cells/ml of Escherichia coli (E. coli) (strain DH10B) suspended in 50 ml of 1 × PBS using a 25 mm paintbrush (Saidi-Mehrabad et al, 2020). The E. coli strain used in our contamination monitoring procedure harbored a mNeonGreen-expressing and ampicillinresistant pBAD/His B vector, which allowed us to trace the contamination at a much smaller levels via direct PCR targeting the vector or macro-photography of the glowing mNeonGreen protein under 470 nm wavelength (Saidi-Mehrabad et al, 2020). All rectangular pieces were decontaminated by the combination of bleach washing and scraping under sterilize conditions designed for ancient DNA analysis in a class 1000 clean lab with no history of sample processing (Saidi-Mehrabad et al, 2020).…”

“…The E. coli strain used in our contamination monitoring procedure harbored a mNeonGreen-expressing and ampicillinresistant pBAD/His B vector, which allowed us to trace the contamination at a much smaller levels via direct PCR targeting the vector or macro-photography of the glowing mNeonGreen protein under 470 nm wavelength (Saidi-Mehrabad et al, 2020). All rectangular pieces were decontaminated by the combination of bleach washing and scraping under sterilize conditions designed for ancient DNA analysis in a class 1000 clean lab with no history of sample processing (Saidi-Mehrabad et al, 2020). Genomic DNA was extracted from a total of ∼10 g of homogenized thawed soil at room temperature from each 6 cm subsection using a modified ZymoBIOMICS DNA Microprep kit (Zymo Research, United States) protocol (Saidi-Mehrabad et al, 2020).…”

“…All rectangular pieces were decontaminated by the combination of bleach washing and scraping under sterilize conditions designed for ancient DNA analysis in a class 1000 clean lab with no history of sample processing (Saidi-Mehrabad et al, 2020). Genomic DNA was extracted from a total of ∼10 g of homogenized thawed soil at room temperature from each 6 cm subsection using a modified ZymoBIOMICS DNA Microprep kit (Zymo Research, United States) protocol (Saidi-Mehrabad et al, 2020). DNA concentration was measured with a Qubit dsDNA HS Assay Kit according to manufacturer's protocol (Thermofisher Scientific, Canada).…”