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.