Sampling and storing
We strongly recommend wearing disposable gloves during sampling to avoid contaminating samples with skin or forward microbiota. To reduce the risk of cross-contamination between independent samples in the field, sampling tools should be replaced or sterilized between sampling events with oxidizing agents (e.g., bleach; Fischer et al., 2016) or flame but not with alcohols, as the latter do not denature DNA. Samples should be collected into clean containers such as paper bags (leaf and fruiting body material), plastic bags (roots, soil and sediments) or screw-cap vessels (soil, water and sediments). It is recommended to sample in the field during dry weather to avoid contamination by water from rain and wet gloves. To enable removal of site or sampling material contaminants a posteriori, it is also recommended to include field controls (e.g., empty tubes left opened at the site or extraction of sample storage buffer) in the experiment (Zinger et al., 2019a). Finally, it is important to limit the biological activity within samples post-harvest (i.e., growth of fast-growing molds), which can be done by maintaining the samples at cold temperature during transport.
To obtain good quality DNA, the best option is to either extract DNA right after sampling whenever possible or to rapidly freeze the collected materials in liquid nitrogen and maintain them at an ultra-cold temperature (-80 °C; U’ren et al., 2014). Pooled subsamples should be well mixed before freezing, because it may subsequently be difficult to homogenize frozen material, which could lead to some subsamples effectively being excluded from DNA extraction. When freezing, it is important to avoid thawing, which may lead to sample spoilage and significant changes in the detected communities (Anslan et al., 2021, Clasen et al., 2020). Long-term storage (2-4 weeks) at 4 °C may alter soil fungal diversity (Delavaux et al., 2020) and promote proliferation of molds (Clasen et al., 2020). Rapid drying methods such as freeze drying and cabinet drying are alternatives to freezing to prevent DNA degradation (Castano et al., 2016). Rapid drying can also be conducted under e.g., the windscreen of a car (at < 40 °C) if a laboratory is inaccessible. Drying with silica gel is a viable option for samples of a few grams (but see Guerrieri et al., 2021 for larger amounts) and will also work well for plant material such as thin leaves and fine roots. Conversely, and importantly, liquid preservatives such as cetyltrimethylammonium bromide (CTAB), ethanol, and specific DNA/RNA preservation solutions perform poorly for above-gram samples (e.g., Delavaux et al., 2020; Zaiko et al., 2021). Nevertheless, Longmire buffer (100 mm Tris, 100 mm ethylenediaminetetraacetic acid, 10 mm NaCl, 0.5% sodium dodecyl sulphate, 0.2% sodium azide; 1:1 vol/vol) works well for sediment and water samples, in which DNA otherwise would degrade very rapidly (Kumar et al., 2020). Dried material and samples fixed in buffers can be kept in the dark at room temperature. Samples can be stored long-term (decades) if kept air-tight in the dark and at constant temperature (Wang et al., 2021). It is also essential to store DNA samples, preferably in frozen or lyophilised form, for potential subsequent quality check, re-analyses for other research purposes or simply for a re-analysis using more sophisticated HTS methods in temporal studies (Jarman et al., 2018). Drying or lyophilisation is essential for pooled samples of coarse fragmented materials (e.g., wood chips and plant litter) which require grinding for adequate mixing.