Common methods for fecal sample storage in field studies yield consistent signatures of individual identity in microbiome sequencing data

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.

Variations in the microbiome due to storage preservatives are not large enough to obscure variations due to factors such as host population, host species, body site, and captivity

“…While there were no significant effects of preservatives on microbial community evenness, we observed significant differences in the microbial community composition and richness due to preservatives. These results are in accordance with our hypothesis that storing identical samples in different preservatives may result in differences in the microbial community composition and richness of the samples and are also consistent with previous reports of both significant (Blekhman et al, ; Choo et al, ; Hale et al, ; Song et al, ; Vlčková et al, ) and nonsignificant (Dominianni, Wu, Hayes, & Ahn, ; Voigt et al, ; Wu et al, ) differences in the microbiota composition related to preservatives. This is possibly a consequence of preservatives on DNA yield and purity, which may result in complications during DNA amplification, sequencing and ultimately to microbiota composition and structure (Hale et al, ; Nsubuga et al, ; Taberlet, Waits, & Luikart, ; Vlčková et al, ).…”

“…This indicates that the effects of these biological factors substantially exceed effects of preservative, making it possible to still see the biological variation even in the presence of technical variation on the microbial community composition and diversity. These results comport with previous studies reporting far smaller preservative differences compared to individual (Blekhman et al, ; Song et al, ; Voigt et al, ), temporal (Voigt et al, ), and host species (Song et al, ) differences. The significantly higher microbial community richness in P. edwardsi compared with V. variegata appears to be driven mainly by presence/absence of microbial taxa (observed OTUs) as opposed to relative abundance which possibly explains the higher number of differentially abundant microbial taxa in the P. edwardsi samples.…”

Consensus on standard biobanking of gut microbiota

“…Appropriate measures should be taken to treat the items used for sample collection and to eliminate potential nucleic acid contamination (eg, ultraviolet irradiation and selective enzymatic degradation). A blank control should always be set up during the collection and processing steps . Blood should be collected by venipuncture.…”

“…A blank control should always be set up during the collection and processing steps. 22,[24][25][26][27][28][29][30][31][32][33][35][36][37][38][39][40][41][42][43] Blood should be collected by venipuncture. The median cubital vein is recommended due to the low risk of contamination.…”

Integrating tumor genomics into studies of the microbiome in colorectal cancer

“…[7][8][9][10][11] Potential sources of cross-study differences can be variation in sample collection, DNA extraction, sequencing target region, sequencing technology used, as well as a wide array of other technical differences. [12][13][14][15][16] Considering recent evidence that host genetic variation is correlated with the composition of the microbiome, [17][18][19] another potential source of variability is the heterogeneity of tumors, and especially the genomic mutational profiles of the tumors. In our recent study, we assessed the effect of tumor mutational profiles on the microbiota in the tumor microenvironment.…”