Author Interview: Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome

Relative abundance of bacterial families identified in fecal samples collected from feral horses on Sable Island, Nova Scotia, Canada, using 16S rRNA sequencing.

PeerJ talked to Stefan Gavriliuc about the recently published article “Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome“. Stefan is pursuing an MSc at the University of Calgary.

Can you tell us a little about yourself and your area of research?
The work described in the PeerJ publication was conducted during my undergraduate degree in bioinformatics at the University of Calgary. I am now pursuing an MSc in the same laboratory where I am focusing on the genetic basis of fitness-related traits in Sable Island horses. I am particularly interested in variation in resistance to parasite infection.

Alpha diversity (richness (A), Choa1 (B) and Shannon indices (C)) of 16S V3–V4 Amplicon Sequence Variants (ASV) for paired equine fecal samples (aliquots) stored at −20 °C and −80 °C for 4 years.

Can you briefly explain the research in your latest PeerJ publication “Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome“?
There are many factors that can affect the microbiome community structure from collecting the sample to analyzing the sequence data. If samples are not immediately processed, storage conditions become important and the consensus is to freeze samples at -80°C. As a result – and as many researchers are likely familiar with – this storage space comes at a premium. We wanted to test whether freezing samples at a more accessible temperature, -20°C, could recover the same bacterial communities as that of those at -80°C. Using 16S amplicon sequencing on technical replicates of equine feces, we saw that there was little variation between bacterial communities using traditional measures for evaluating diversity within and among samples.

What do you hope people take away from this research?
We hope that this work will increase the accessibility of conducting studies on host bacterial microbiomes in places where storage at -80C is hard to come by. In addition, this research was a part of a larger project being conducted on microbiome diversity & function in Sable Island horses, where we are hoping to describe the factors behind microbiome community assembly in a social, free-living host animal. We recently published a first look at these factors where we saw that spatial and social variables were among the primary driving forces in community assembly. We hope to continue generating more longitudinal genetic data and we seek to further characterize the microbiome of these horses using other methods such as shallow or deep shotgun metagenomics to improve our understanding of these complex dynamics.

Non-metric multidimensional scaling (NMDS) of equine gut bacterial communities inferred from fecal samples stored at −20 °C and −80 °C.

What is next for your research?
My current research centers on generating host genomic information for Sable Island horses and contrast this data to measures of parasite burden to identify whether there are genetic sites are associated with a propensity or resistance against parasite infection. Down the road, we are also interested in applying a similar approach to the microbiome to see whether host genetics explain variation in the community composition of the gut microbiota.

Would you recommend PeerJ to your colleagues?

I would recommend PeerJ because of their focus on scientific soundness rather than impact or novelty. A lot of compelling research may go unpublished when evaluating work based on perceived interest. We also received thoughtful feedback during the revision process which was helpful for completing the manuscript.

You can read more PeerJ author interviews here.