Multi ‘Omics Analysis of Intestinal Tissue in Ankylosing Spondylitis Identifies Alterations in the Tryptophan Metabolism Pathway

Intestinal microbial dysbiosis, intestinal inflammation, and Th17 immunity are all linked to the pathophysiology of spondyloarthritis (SpA); however, the mechanisms linking them remain unknown. One potential hypothesis suggests that the dysbiotic gut microbiome as a whole produces metabolites that influence human immune cells. To identify potential disease-relevant, microbiome-produced metabolites, we performed metabolomics screening and shotgun metagenomics on paired colon biopsies and fecal samples, respectively, from subjects with axial SpA (axSpA, N=21), Crohn’s disease (CD, N=27), and Crohn’s-axSpA overlap (CD-axSpA, N=12), as well as controls (HC, N=24). Using LC-MS based metabolomics of 4 non-inflamed pinch biopsies of the distal colon from subjects, we identified significant alterations in tryptophan pathway metabolites, including an expansion of indole-3-acetate (IAA) in axSpA and CD-axSpA compared to HC and CD and indole-3-acetaldehyde (I3Ald) in axSpA and CD-axSpA but not CD compared to HC, suggesting possible specificity to the development of axSpA. We then performed shotgun metagenomics of fecal samples to characterize gut microbial dysbiosis across these disease states. In spite of no significant differences in alpha-diversity among the 4 groups, our results confirmed differences in gene abundances of numerous enzymes involved in tryptophan metabolism. Specifically, gene abundance of indolepyruvate decarboxylase, which generates IAA and I3Ald, was significantly elevated in individuals with axSpA while gene abundances in HC demonstrated a propensity towards tryptophan synthesis. Such genetic changes were not observed in CD, again suggesting disease specificity for axSpA. Given the emerging role of tryptophan and its metabolites in immune function, altogether these data indicate that tryptophan metabolism into I3Ald and then IAA is one mechanism by which the gut microbiome potentially influences the development of axSpA.

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An interventional Soylent diet increases the Bacteroidetes to Firmicutes ratio in human gut microbiome communities: a randomized controlled trial

10.1101/200881 ◽

2017 ◽

Cited By ~ 1

Author(s):

Ryan H. Hsu ◽

Dylan M. McCormick ◽

Mitchell J. Seitz ◽

Lauren M. Lui ◽

Harneet S. Rishi ◽

...

Keyword(s):

Gut Microbiome ◽

Intestinal Inflammation ◽

Controlled Trial ◽

Alpha Diversity ◽

Diet Group ◽

Positive Health ◽

Unifrac Distance ◽

Liquid Meal ◽

Regular Diet ◽

Rdna Sequencing

AbstractOur knowledge of the relationship between the gut microbiome and health has rapidly expanded in recent years. Diet has been shown to have causative effects on microbiome composition, which can have subsequent implications on health. Soylent 2.0 is a liquid meal replacement drink that satisfies nearly 20% of all recommended daily intakes per serving. This study aims to characterize the changes in gut microbiota composition resulting from a short-term Soylent diet. Fourteen participants were separated into two groups: 5 in the regular diet group and 9 in the Soylent diet group. The regular diet group maintained a diet closely resembling self-reported regular diets. The Soylent diet group underwent three dietary phases: A) a regular diet for 2 days, B) a Soylent-only diet (five servings of Soylent daily and water as needed) for 4 days, and C) a regular diet for 4 days. Daily logs self-reporting diet, Bristol stool ratings, and any abdominal discomfort were electronically submitted. Eight fecal samples per participant were collected using fecal sampling kits, which were subsequently sent to uBiome, Inc. for sample processing and V4 16S rDNA sequencing. Reads were clustered into operational taxonomic units (OTUs) and taxonomically identified against the GreenGenes 16S database. We find that an individual’s alpha-diversity is not significantly altered during a Soylent-only diet. In addition, principal coordinate analysis using the unweighted UniFrac distance metric shows samples cluster strongly by individual and not by dietary phase. Among Soylent dieters, we find a significant increase in the ratio of Bacteroidetes to Firmicutes abundance, which is associated with several positive health outcomes, including reduced risks of obesity and intestinal inflammation.

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The Oral and Gut Bacterial Microbiomes: Similarities, Differences, and Connections

Biological Research For Nursing ◽

10.1177/1099800420941606 ◽

2020 ◽

Vol 23 (1) ◽

pp. 7-20

Author(s):

Katherine A. Maki ◽

Narjis Kazmi ◽

Jennifer J. Barb ◽

Nancy Ames

Keyword(s):

Gut Microbiome ◽

Alpha Diversity ◽

Oral Microbiome ◽

Taxonomic Resolution ◽

Future Research ◽

Sequencing Analysis ◽

Systemic Diseases ◽

Shotgun Metagenomics ◽

Healthy Humans ◽

Microbiome Research

Background: The oral cavity is associated with local and systemic diseases, although oral samples are not as commonly studied as fecal samples in microbiome research. There is a gap in understanding between the similarities and differences in oral and gut microbiomes and how they may influence each other. Methods: A scoping literature review was conducted comparing oral and gut microbiome communities in healthy humans. Results: Ten manuscripts met inclusion criteria and were examined. The oral microbiome sites demonstrated great variance in differential bacterial abundance and the oral microbiome had higher alpha diversity as compared to the gut microbiome. Studies using 16S rRNA sequencing analysis resulted in overall community differences between the oral and gut microbiomes when beta diversity was analyzed. Shotgun metagenomics sequencing increased taxonomic resolution to strain level (intraspecies) and demonstrated a greater percentage of shared taxonomy and oral bacterial translocation to the gut microbiome community. Discussion: The oral and gut microbiome bacterial communities may be more similar than earlier research has suggested, when species strain is analyzed through shotgun metagenomics sequencing. The association between oral health and systemic diseases has been widely reported but many mechanisms underlying this relationship are unknown. Although future research is needed, the oral microbiome may be a novel interventional target through its downstream effects on the gut microbiome. As nurse scientists are experts in symptom characterization and phenotyping of patients, they are also well posed to lead research on the connection of the oral microbiome to the gut microbiome in health and disease.

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Impacts of Aging and Blackcurrant Supplementation on the Gut Microbiome Profile of Female Mice (P20-031-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz040.p20-031-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Sang Gil Lee ◽

Cao Lei ◽

Melissa Melough ◽

Junichi Sakaki ◽

Kendra Maas ◽

...

Keyword(s):

Relative Abundance ◽

Gut Microbiome ◽

Health Benefits ◽

Alpha Diversity ◽

Young Female ◽

Rrna Gene ◽

Fecal Samples ◽

Female Mice ◽

Aged Mice ◽

Young Mice

Abstract Objectives Blackcurrant, an anthocyanin-rich berry, has multiple health benefits. The purpose of this study was to examine the impacts of blackcurrant supplementation and aging on gut bacterial communities in female mice. Methods Three-month and 18-month old female mice were provided standard chow diets with or without anthocyanin-rich blackcurrant extract (BC) (1% w/w) for four months. Upon study completion, fecal samples were collected directly from the animals’ colons. Microbiome DNA was extracted from the fecal samples and the V3-V4 regions of their 16S rRNA gene were amplified and sequenced using Results Taxonomic analysis showed a significantly decrease in alpha diversity in aged female mice, compared to young counterparts. BC consumption did not alter the alpha diversity in either young or aged mice compared to control diets. For beta diversity, we observed the clustering was associated with age but not diet. The phylogenic abundance analysis showed that the relative abundance of several phyla, including Firmicutes, Bacteroidetes, Cyanobacteria, Proteobacteria, and Tenericutes was higher in aged compared to young mice. Among them, the abundance of Firmicutes was downregulated by BC in the young but not the aged mice. The abundance of Bacteroidetes was increased by BC in both the young and the aged groups. Noticeably, Verrucomicrobia was the only phylum whose relative abundance was upregulated in the aged female mice compared to the young mice. Meanwhile, its relative abundance in the aged group was suppressed by BC. Interestingly, Desulfovibrio, which is the most representative sulfate-reducing genus, was detectable only in young female mice, and BC increased its relative abundance. Conclusions Our results characterized the gut microbiome compositions in young and aged female mice, and indicated that the gut microbiome of young and aged female mice responded differently to four month BC administration. Through additional research, the microbial alterations observed in this study should be further investigated to inform our understanding of the effect of BC on the gut microbiome, the possible health benefits related to these changes, and the differing effects of BC supplementation across populations. Funding Sources This study was supported by the USDA NIFA Seed Grant (#2016-67018-24492) and the University of Connecticut Foundation Esperance Funds to Dr. Ock K. Chun. We thank the National Institute on Aging for providing aged mice for the project and Just the Berries Ltd. for providing the blackcurrant extract.

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A260 THE LACK OF CHROMOGRANIN-A MODIFIES THE GUT MICROBIOTA COMPOSITION AND REGULATES EXPERIMENTAL COLONIC INFLAMMATION

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwz047.259 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. 137-138

Author(s):

N Eissa ◽

A Diarra ◽

H Hussein ◽

C N Bernstein ◽

J Ghia

Keyword(s):

Gut Microbiota ◽

Gut Microbiome ◽

Chromogranin A ◽

Bacterial Community Composition ◽

Alpha Diversity ◽

Lactobacillus Species ◽

Wild Type ◽

Microbial Composition ◽

Colonic Inflammation ◽

Microbial Dysbiosis

Abstract Background Ulcerative colitis (UC)is characterized by distinct changes in the gut microbiome and elevated chromogranin-A (CHGA) level, which seem to be a relevant pathogenetic mechanism.CHGA, a prohormone produced by enterochromaffin (EC) cells and cleaved into several bioactive peptides, regulates experimental colonic inflammation. In the rodent, intra-rectal infusion of catestatin, a Chga-derived peptide, alters the distal colonic microbial composition. However, the interplay between CHGA, as a pro-hormone, and the gut microbiome remains elusive. Aims in homoeostatic and pathophysiologic conditions, we investigated the functional consequences of the lack of Chgaon the distal colonic microbiota. Methods Acute colitis (5 % dextran sulfate sodium [DSS], 5 days) was induced in Chga-C57BL/6-deficient (Chga-/-) and wild-type (Chga+/+)mice. Feces and mucosa-associated microbiota (MAM) samples were collected and the V4 region of 16s rRNA was subjected to Miseq Illumina sequencing. Alpha diversity was calculated using Shannon’s diversity index. OTU abundances were summarized using the Bray-Curtis index and non-metric multidimensional scaling (NMDS) analysis to visualize microbiome similarities and a permutational analysis of variance (PERMANOVA) to test the significance of groups were performed respectively. Results In non-colitic homoeostatic condition, the absence of Chga (Chga-/) significantly increased the bacterial richness and modified the bacterial community composition at the genera level between the groups, represented by increased abundance of Lactobacillus species and reduced abundance of Helicobacter& Oscillospira species compared to Chga+/+mice in fecal and colonic MAM. Moreover, the absence of Chga (Chga-/-) resulted in a significant change in the alpha-diversity of fecal and colonic MAM compared to Chga+/+mice. DSS induced-colitis resulted in a significant microbial dysbiosis in Chga+/+mice, however, deletion of Chgaprotected against DSS-induced colitis and reduced the microbial dysbiosis, reduced the family of Rikenellaceaeand maintained the abundance of Bacteroides species, compared to wild-type (Chga+/+). Conclusions The lack of CHGA regulates the biodiversity and the composition of the colonic gut microbiota suggesting a cross-talk between the EC cell and the microbiome. Therefore, targeting CHGA could provide a novel therapeutic strategy by regulating the gut microbiome in physiological and pathophysiological conditions. Funding Agencies CIHR

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Whole-Genome Metagenomic Analysis of the Gut Microbiome in HIV-1-Infected Individuals on Antiretroviral Therapy

Frontiers in Microbiology ◽

10.3389/fmicb.2021.667718 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiangning Bai ◽

Aswathy Narayanan ◽

Piotr Nowak ◽

Shilpa Ray ◽

Ujjwal Neogi ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Gut Microbiome ◽

Alpha Diversity ◽

16S Rrna Gene Sequencing ◽

Taxonomic Resolution ◽

Rrna Gene ◽

Human Gut ◽

Shotgun Metagenomics ◽

Human Gut Microbiome ◽

Hiv 1

Gut microbiome plays a significant role in HIV-1 immunopathogenesis and HIV-1-associated complications. Previous studies have mostly been based on 16S rRNA gene sequencing, which is limited in taxonomic resolution at the genus level and inferred functionality. Herein, we performed a deep shotgun metagenomics study with the aim to obtain a more precise landscape of gut microbiome dysbiosis in HIV-1 infection. A reduced tendency of alpha diversity and significantly higher beta diversity were found in HIV-1-infected individuals on antiretroviral therapy (ART) compared to HIV-1-negative controls. Several species, such as Streptococcus anginosus, Actinomyces odontolyticus, and Rothia mucilaginosa, were significantly enriched in the HIV-1-ART group. Correlations were observed between the degree of immunodeficiency and gut microbiome in terms of microbiota composition and metabolic pathways. Furthermore, microbial shift in HIV-1-infected individuals was found to be associated with changes in microbial virulome and resistome. From the perspective of methodological evaluations, our study showed that different DNA extraction protocols significantly affect the genomic DNA quantity and quality. Moreover, whole metagenome sequencing depth affects critically the recovery of microbial genes, including virulome and resistome, while less than 5 million reads per sample is sufficient for taxonomy profiling in human fecal metagenomic samples. These findings advance our understanding of human gut microbiome and their potential associations with HIV-1 infection. The methodological assessment assists in future study design to accurately assess human gut microbiome.

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Gestational insulin resistance is mediated by the gut microbiome-indoleamine 2,3-dioxygenase axis

10.1101/2021.07.21.453234 ◽

2021 ◽

Author(s):

Medha Priyadarshini ◽

Guadalupe Navarro ◽

Derek Reiman ◽

Anukriti Sharma ◽

Kai Xu ◽

...

Keyword(s):

Insulin Resistance ◽

Gut Microbiome ◽

Intestinal Inflammation ◽

Alpha Diversity ◽

Mouse Strains ◽

Indoleamine 2,3 Dioxygenase ◽

Plasma Metabolome ◽

Maternal Metabolism ◽

Nih Swiss ◽

Normal Gestation

Background and aims: Normal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM maternal metabolism interaction. Methods: The GM and plasma metabolome of CD1, NIH Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT). Results: Significant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post-pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy. Conclusions: GM changes accompanying pregnancy shift IDO1 dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

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CBMT-40. THE RELATIONSHIP BETWEEN GLIOMA AND THE GUT-BRAIN AXIS

Neuro-Oncology ◽

10.1093/neuonc/noz175.162 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi41-vi42

Author(s):

Anthony Patrizz ◽

Soheil Zorofchain ◽

Gabriella Hines ◽

Takeshi Takayasu ◽

Yoshihiro Otani ◽

...

Keyword(s):

Beta Diversity ◽

Alpha Diversity ◽

Fecal Samples ◽

Fecal Microbiome ◽

Therapeutic Modalities ◽

Microbial Dysbiosis ◽

16S Rna ◽

Hydroxyindoleacetic Acid ◽

Immune Oncology ◽

The Relationship

Abstract Recent studies demonstrate the potential role of the microbiome in immune-oncology, revealing specific microbial taxa can augment the effects of various therapeutic modalities against tumors. Gut dysbiosis, a disequilibrium in the host’s bacterial ecosystem, can potentially lead to overrepresentation of some bacteria and favor chronic inflammation and immunosuppression. However, the effects of microbial dysbiosis on non-gastrointestinal cancers in particular gliomas are unknown. Here, we explored the effects of glioma and Temozolomide (TMZ) on the fecal microbiome (FM) in mice (n=24) and FM and metabolome in humans (n=40). Aged C57/B6 mice were implanted with Gl261 tumor cells or vehicle and were assigned to one of the following treatment (oral) groups: vehicle, 5mg/kg TMZ or 25mg/kg TMZ beginning 14 days after surgery for 3-weeks following a 5 day on/2 day off treatment. Fecal samples were collected prior to surgery, at treatment initiation and weekly thereafter until sacrifice and sequenced for 16s RNA. Fecal samples were collected from humans with newly diagnosed glioma before resection, chemoradiation, and after chemoradiation (16s RNA, metabolomic, neurotransmitter analysis). In mice, FM beta diversity was significantly altered with glioma (p=0.003) while the alpha diversity remained unchanged. At a genus and family level analysis the relative abundance of Bacteroides (p=0.01) and Bacteroidaceae (p=0.02) was increased. Beta diversity of mice receiving 5mg/kg TMZ changed from baseline (p=0.02). Collectively, this suggests that glioma alters the FM, to what consequence remains to be explored. Alpha (Observed OTUs, p=0.029) and beta diversity (p=0.034) differences in mice correlated with survival (< 25 - >25 days). In humans, norepinephrine and 5-hydroxyindoleacetic acid were significantly lower in glioma patients at diagnosis compared to controls. Our findings demonstrate for the first time the relationship between glioma and the gut-brain axis. Understanding alterations in the FM in glioma patients may allow novel interventions and should be further investigated.

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Glioma and temozolomide induced alterations in gut microbiome

Scientific Reports ◽

10.1038/s41598-020-77919-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Anthony Patrizz ◽

Antonio Dono ◽

Soheil Zorofchian ◽

Gabriella Hines ◽

Takeshi Takayasu ◽

...

Keyword(s):

Mouse Model ◽

Gut Microbiome ◽

Healthy Controls ◽

Fecal Samples ◽

Fecal Microbiome ◽

Therapeutic Modalities ◽

Microbial Dysbiosis ◽

Rrna Sequencing ◽

Before And After ◽

Immune Oncology

AbstractThe gut microbiome is fundamental in neurogenesis processes. Alterations in microbial constituents promote inflammation and immunosuppression. Recently, in immune-oncology, specific microbial taxa have been described to enhance the effects of therapeutic modalities. However, the effects of microbial dysbiosis on glioma are still unknown. The aim of this study was to explore the effects of glioma development and Temozolomide (TMZ) on fecal microbiome in mice and humans. C57BL/6 mice were implanted with GL261/Sham and given TMZ/Saline. Fecal samples were collected longitudinally and analyzed by 16S rRNA sequencing. Fecal samples were collected from healthy controls as well as glioma patients at diagnosis, before and after chemoradiation. Compared to healthy controls, mice and glioma patients demonstrated significant differences in beta diversity, Firmicutes/Bacteroides (F/B) ratio, and increase of Verrucomicrobia phylum and Akkermansia genus. These changes were not observed following TMZ in mice. TMZ treatment in the non-tumor bearing mouse-model diminished the F/B ratio, increase Muribaculaceae family and decrease Ruminococcaceae family. Nevertheless, there were no changes in Verrucomicrobia/Akkermansia. Glioma development leads to gut dysbiosis in a mouse-model, which was not observed in the setting of TMZ. These findings seem translational to humans and warrant further study.

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The impact of gut microbial composition on response to neoadjuvant chemotherapy (NACT) in early-stage triple negative breast cancer (TNBC).

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.590 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 590-590

Author(s):

Nour Abuhadra ◽

Chia-Chi Chang ◽

Clinton Yam ◽

Jason B White ◽

Elizabeth Ravenberg ◽

...

Keyword(s):

Gut Microbiome ◽

Early Stage ◽

Univariate Analysis ◽

Alpha Diversity ◽

Microbial Composition ◽

Fecal Microbiota Transplant ◽

Fecal Samples ◽

Response To Chemotherapy ◽

Significant Difference ◽

The Impact

590 Background: The impact of gut microbiome on tumor biology, progression and response to immunotherapy has been shown across cancer types. However, there is little known about the impact of gut microbial composition on response to chemotherapy. We have previously shown that the gut microbiome remains unaltered during NACT in a cohort of 32 patients. Here we investigate the association between gut microbiome and response to NACT in a larger cohort of early-stage TNBC. Methods: Longitudinal fecal samples were collected from 85 patients with newly-diagnosed, early-stage TNBC patients enrolled in the ARTEMIS trial (NCT02276443). Patients all received standard NACT with adriamycin/cyclophosphamide (AC); volumetric change was assessed using ultrasound and patients with < 70% volumetric reduction (VR) after 4 cycles of AC were recommended to receive targeted therapy in addition to standard NACT to improve response rates. We performed 16S sequencing on bacterial genomic DNA extracted from 85 pre-AC fecal samples using the 2x250 bp paired-end read protocol. Quality-filtered sequences were clustered into Operational Taxonomic Units and classified using Mothur method with the Silva database version 138. For differential taxa-based univariate analysis, abundant microbiome taxa at species, genus, family, class, and order levels were analyzed using DESeq2 after logit transformation. Alpha-diversity indices within group categories were calculated using phyloseq. Microbial alpha diversity (within-sample diversity) was measured by Simpson's reciprocal index. β-diversity was measured using weighted UniFrac distances between the groups. The association between microbiota abundance and pathologic complete response (pCR) or residual disease (RD) was assessed using DESeq2 analysis. Results: Pre-AC fecal samples from 85 patients were available for analysis. Amongst them, there were 46 patients with pCR and 39 patients with RD. There was no significant difference in alpha diversity (p = 0.8) or beta-diversity (p = 0.7) between the pCR and RD groups. However, relative to patients with RD, the gut microbiome in patients with pCR was enriched for the Bifidobacterium longum species (p = 0.03). The gut microbiome in patients with RD was enriched for Lachnospiraceae (p = 0.03) at the genus level and the Bacteroides thetaiotaomicron species (p = 0.02). Conclusions: We have demonstrated significant differences in the gut microbial composition in patients with pCR as compared to patients with RD. Further investigation in larger studies is needed to support therapeutic exploration of gut microbiome modulation in TNBC patients receiving chemotherapy such as probiotic supplementation or fecal microbiota transplant.

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

10.1101/038844 ◽

2016 ◽

Author(s):

Ran Blekhman ◽

Karen Tang ◽

Elizabeth Archie ◽

Luis Barreiro ◽

Zachary Johnson ◽

...

Keyword(s):

Gut Microbiome ◽

Alpha Diversity ◽

Field Studies ◽

Interindividual Variation ◽

Individual Identity ◽

Sample Storage ◽

Sequencing Data ◽

Fecal Samples ◽

Storage Effects ◽

Storage Methods

Field studies of wild vertebrates are frequently associated with extensive collections of banked fecal samples, which are often collected from known individuals and sometimes also sampled longitudinally across time. Such collections represent unique resources for understanding ecological, behavioral, and phylogenetic effects on the gut microbiome, especially for species of particular conservation concern. However, we do not understand whether sample storage methods confound the ability to investigate interindividual variation in gut microbiome profiles. This uncertainty arises in part because comparisons across storage methods to date generally include only a few (≤5) individuals, or analyze pooled samples. Here, we used n=52 samples from 13 rhesus macaque individuals to compare immediate freezing, the gold standard of preservation, to three methods commonly used in vertebrate field studies: storage in ethanol, lyophilization following ethanol storage, 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.

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