Treated like dirt: Robust forensic and ecological inferences from soil eDNA after challenging sample storage

Abstract

Biodiversity of soil microbiota is routinely assessed with environmental DNA-based methods, among which amplification and massive parallel sequencing of marker genes (eDNA metabarcoding) is the most common. Soil microbiota may for example be investigated in relation to biodiversity research or as a tool in forensic investigations. After sampling, the taxonomic composition of soil biotic communities may change. In order to minimize community changes after sampling, it is desirable to reduce biological activity, e.g. by freezing immediately after sampling. However, this may be impossible due to remoteness of study sites or, in forensic cases, where soil has been attached to a questioned item for protracted periods of time. Here we investigated the effect of storage duration and conditions on the assessment of the soil biota with eDNA metabarcoding. We extracted eDNA from freshly collected soil samples and again from the same samples after storage under contrasting temperature conditions. We used five different primer sets targeting bacteria, fungi, protists (cercozoans), general eukaryotes, and plants. For these groups, we quantified differences in richness, evenness and community composition. Subsequently, we tested whether we could correctly infer habitat type and original sample identity after storage using a large reference dataset. We found increased community composition differences with extended storage time and with higher storage temperature. However, for samples stored less than 28 days at a maximum of 20{degrees}C, changes were generally insignificant. Classification models could successfully assign most stored samples to their exact location of origin and correct habitat type even after weeks of storage. Even samples showing larger compositional changes generally retained the original sample as the best match (relative similarity). Our results show that for most biodiversity and forensic applications, storage of samples for days and even several weeks may not be a problem, if storage temperature does not exceed 20{degrees}C. Even after suboptimal storage conditions, significant patterns can be reproduced.