Abstract
Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples—unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. Here, we extend previous work on storage methods for gut microbiome samples by comparing immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: lyophilization, storage in ethanol, and storage in RNAlater. We found that the signature of individual identity consistently outweighed storage effects: alpha diversity and beta diversity measures were significantly correlated across methods, and while samples often clustered by donor, they never clustered by storage method. Provided that all analyzed samples are stored the same way, banked fecal samples therefore appear highly suitable for investigating variation in gut microbiota. Our results open the door to a much-expanded perspective on variation in the gut microbiome across species and ecological contexts.
Highlights
Versus individual identity, as the latter captures the biological variation generally of most interest to researchers
For each individual in the study, we collected a fresh stool sample and divided it into four aliquots (n = 52 samples; Table S1). These were stored for 2–6 days prior to DNA extraction via: 1) immediate freezing at −20 °C; 2) immersion in absolute ethanol; 3) immersion in the preservative RNAlater; or 4) immersion in ethanol followed by lyophilization to powder
In our case, lyophilized samples exhibited lower Shannon’s Diversity Index (SDI) values relative to other conditions (Tukey’s HSD: p between 7.6 × 10−5 and 0.063), and samples stored in RNAlater exhibited somewhat higher values, this comparison was only significant in comparison to the lyophilized condition (p = 7.6 × 10−5; Fig. 1a; Table S2)
Summary
Versus individual identity, as the latter captures the biological variation generally of most interest to researchers (whereas storage-driven variance is purely technical). Storage methods often explain substantial variation in microbiome composition when all other sources of variance are controlled[2,3], the degree to which they confound other analyses depends on their importance relative to the effects of biologically interesting variation (interindividual, temporal, and environmental). We evaluated individual identity versus technical storage effects on both alpha diversity and beta diversity estimates from high sequencing-depth 16S rRNA profiles. This study design provides a conservative estimate of the contribution of individual identity relative to technical variance, as all animals were sampled in standardized housing, dietary, and social group conditions
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