Microeukaryotes in metagenomic survey of ancient Siberian permafrost

Study Extent

The samples were collected in the field and stored constantly frozen during all periods of transportation and processing. In the lab, a part of each sample was used to isolate protist strains. The remaining part has never been melted and is stored at −18 °C. The isolation was done in sterile conditions. Obtained samples were cloned and maintained as bacterized or axenic cultures on plastic or agar with liquid overlay.

Sampling

The drilling was performed using a mobile drilling rig (core-drilling machine) UKB-12/25 (V.V. Vorovsky Machine-Building Plant, Moscow, Russia) operated without flushing and blowing. Flushing and blowing were shown to cause contamination of the cores by modern soil microorganisms. Cores were removed each 30—70 cm of the drilling. The core diameters were 115 to 75 mm, depending on the well depth (the deeper the well the smaller). A removed core was enveloped in a one-centimeter-thick coat of half-melted cuttings. This coat was removed with a knife, showing a completely frozen inner part. After a short lithological and glaciological description of the sediments, the core was passed to a clean field lab organized in a tent. Operations in the lab were conducted behind a gas-fired burner using disposable materials and gloves, following general microbiological practice. In the lab, the core was shaved with a sterile scalpel so that an approximately five mm outermost layer was removed. This remaining core was four to six cm in diameter, depending on the initial value. Immediately after shaving, the core was put into a sterile plastic bag and placed in a portable freezer, a cave dug into an ice wedge or an empty borehole used as a freezer. In total, the “outdoor” stage of the process lasted five to ten minutes, depending on the current well depth. The “indoor” lab stage generally took around five minutes. All collected cores were kept at negative temperatures during the whole period of transportation to the stationary lab.

Quality Control

During the development of the permafrost microbiological sampling technique, several tests for contamination of the core interior have been established at different phases of sampling and storage. Gilichinskiy et al. (2010) and Shi et al. (1997) used Serratia marcescens cultures (Bacteria) producing easy-to-notice red colonies. A drilling barrel was covered with a culture suspension 2 h prior to drilling. In a parallel experiment, frozen samples were seeded with the same suspension already in the lab and left intact at negative temperature for several hours to several months. In both experiments, the distribution of S. marcescens cells in a core has been investigated during the sample processing. In all tests, bacteria have been found exclusively in the outer layer and never inside the core.

Method steps

1. Field sampling The drilling was performed using a mobile drilling rig (core-drilling machine) UKB-12/25 (V.V. Vorovsky Machine-Building Plant, Moscow, Russia) operated without flushing and blowing. Cores were removed each 30—70 cm of the drilling. The core diameters were 115 to 75 mm, depending on the well depth (the deeper the well the smaller). A removed core was enveloped in a one-centimeter-thick coat of half-melted cuttings. This coat was removed with a knife, showing a completely frozen inner part. After a short lithological and glaciological description of the sediments, the core was passed to a clean field lab organized in a tent. Operations in the lab were conducted behind a gas-fired burner using disposable materials and gloves, following general microbiological practice. In the lab, the core was shaved with a sterile scalpel so that an approximately five mm outermost layer was removed. This remaining core was four to six cm in diameter, depending on the initial value. Immediately after shaving, the core was put into a sterile plastic bag and placed in a portable freezer, a cave dug into an ice wedge or an empty borehole used as a freezer. In total, the “outdoor” stage of the process lasted five to ten minutes, depending on the current well depth. The “indoor” lab stage generally took around five minutes. All collected cores were kept at negative temperatures during the whole period of transportation to the stationary lab.
2. DNA isolation In the laboratory, material from the inner part of the permafrost cores was subsampled aseptically for DNA isolation. The gDNA was extracted from eight replicates of ∼ 0.5 g each randomly taken from ∼ 50 g of permafrost core collected at corresponding depth (Fig. 1) using the PowerSoil® DNA Extraction Kit (MO BIO Laboratories Inc., USA). Due to low yield (3–6 ng μL −1 ), gDNAs from eight replicates were combined, then purified and concentrated using Genomic DNA Clean and Concentrator® Kit (Zymo Research Corporation, USA).
3. Library preparation and sequencing The gDNA sequencing libraries were prepared using NEBNext® reagents (New England BioLabs Inc., USA), according to protocol recommended by the manufacturer, having an estimated peak insert size of 150 nt. Metagenome sequencing was performed at the CRG Genomics Core Facility (Centre for Genomic Regulation, Barcelona, Spain) on an Illumina HiSeq 2000 TM machine using Flow Cell v3 with TruSeq SBS v3 reagents and a 2×100 cycle sequencing protocol.
4. MG-RAST annotation IC4 and IC8 Raw sequencing data (i.e., 19.8 Gb, representing 143.7 M sequences with an average length of 138 bp for IC4, and 19.7 Gb, representing 131.7 M sequences with an average length of 150 bp for IC8) were uploaded to the MG-RAST server (Meyer et al., 2008) for gene calling and annotation under ID 4 606 864.3 for IC4 and 4 606 865.3 for IC8. A total of 6.6% (IC4) and 3.4% (IC8) of the sequences failed to pass the quality control (QC) pipeline, whereas 0.3% of the total sequences in both data sets were assigned to ribosomal RNA genes. The taxonomic assignment of Illumina reads was performed against M5NR and M5RNA databases with the default parameters. The best-hit classification method was used in both cases for match assessment. IC13 and IC15 To assess the diversity and abundance of eukaryotic microorganisms, we have used sequences of 18S rRNA gene fragments annotated using the Silva database (Quast et al., 2013) with the following parameters: threshold e-value, 1×10-5; minimal identity threshold, 90%; and minimum alignment length, 60 base pairs. We chose the genus as the initial level of taxonomic annotation and identified the taxonomic position of recognized genera following the current systematics of eukaryotes (Adl et al., 2019; Pawlowski, 2014; Smirnov et al., 2011).

Sampling sites span Arctic locations in Siberia from the Gydan Peninsula to Chukotka.