Dataset Information

Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

ABSTRACT:

Background

In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at -80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for saliva) among 51 healthy volunteers.

Methods

Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the 20 most abundant genera, three alpha-diversity metrics, and the first principal coordinates of three beta-diversity matrices.

Results

The subject variability was much higher than the variability introduced by the sample collection type, and storage time. For fecal samples, microbial stability over 2 years was high across collection methods (range, ICCs = 0.70-0.99), except for the samples collected with no additive (range, ICCs = 0.23-0.83). For oral samples, most microbiome diversity measures were stable over time with ICCs above 0.74; however, ICCs for the samples collected with Scope mouthwash were lower for two alpha-diversity measures, Faith's phylogenetic diversity (0.23) and the observed number of operational taxonomic units (0.23).

Conclusions

Fecal and oral samples in most used collection methods are stable for microbiome analyses after 2 years at -80°C, except for fecal samples with no additive.

Impact

This study provides evidence that samples stored for an extended period from prospective studies are useful for microbiome analyses.

SUBMITTER: Zouiouich S

PROVIDER: S-EPMC10498478 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

Zouiouich Semi S Byrd Doratha A DA Hua Xing X Karwa Smriti S Wan Yunhu Y Shi Jianxin J Humphrey Gregory C GC Ackermann Gail L GL Knight Rob R Abnet Christian C CC Vogtmann Emily E Sinha Rashmi R

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 20230301 3

<h4>Background</h4>In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at -80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for ...[more]

PMID: 36649143

Similar Datasets

Project description:Background: Integrated microbiome and metabolomics analyses hold the potential to reveal interactions between host and microbiota in relation to disease risks. However, there are few studies evaluating how field methods influence fecal microbiome characterization and metabolomics profiling. Methods: Five fecal collection methods [immediate freezing at -20°C without preservative, OMNIgene GUT, 95% ethanol, RNAlater, and Flinders Technology Associates (FTA) cards] were used to collect 40 fecal samples from eight healthy volunteers. We performed gut microbiota 16S rRNA sequencing, untargeted metabolomics profiling, and targeted metabolomics focusing on short chained fatty acids (SCFAs). Metrics included α-diversity and β-diversity as well as distributions of predominant phyla. To evaluate the concordance with the "gold standard" immediate freezing, the intraclass correlation coefficients (ICCs) for alternate fecal collection systems were calculated. Correlations between SCFAs and gut microbiota were also examined. Results: The FTA cards had the highest ICCs compared to the immediate freezing method for α-diversity indices (ICCs = 0.96, 0.96, 0.76 for Shannon index, Simpson's Index, Chao-1 Index, respectively), followed by OMNIgene GUT, RNAlater, and 95% ethanol. High ICCs (all >0.88) were observed for all methods for the β-diversity metric. For untargeted metabolomics, in comparison to immediate freezing which detected 621 metabolites at ≥75% detectability level, 95% ethanol showed the largest overlapping set of metabolites (n = 430; 69.2%), followed by FTA cards (n = 330; 53.1%) and OMNIgene GUT (n = 213; 34.3%). Both OMNIgene GUT (ICCs = 0.82, 0.93, 0.64) and FTA cards (ICCs = 0.87, 0.85, 0.54) had acceptable ICCs for the top three predominant SCFAs (butyric acid, propionic acid and acetic acid). Nominally significant correlations between bacterial genera and SCFAs (P < 0.05) were observed in fecal samples collected by different methods. Of note, a high correlation between the genus Blautia (known butyrate producer) and butyric acid was observed for both immediate freezing (r = 0.83) and FTA cards (r = 0.74). Conclusions: Four alternative fecal collection methods are generally comparable with immediate freezing, but there are differences in certain measures of the gut microbiome and fecal metabolome across methods. Choice of method depends on the research interests, simplicity of fecal collection procedures and ease of transportation to the lab, especially for large epidemiological studies.

Project description:Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.

Project description:BackgroundTo initiate fecal and oral collections in prospective cohort studies for microbial analyses, it is essential to understand how field conditions and geographic differences may impact microbial communities. This study aimed to investigate the impact of fecal and oral sample collection methods and room temperature storage on collection samples for studies of the human microbiota.ResultsWe collected fecal and oral samples from participants in two Iranian cohorts located in rural Yazd (n = 46) and urban Gonbad (n = 38) and investigated room temperature stability over 4 days of fecal (RNAlater and fecal occult blood test [FOBT] cards) and comparability of fecal and oral (OMNIgene ORAL kits and Scope mouthwash) collection methods. We calculated interclass correlation coefficients (ICCs) based on 3 alpha and 4 beta diversity metrics and the relative abundance of 3 phyla. After 4 days at room temperature, fecal stability ICCs and ICCs for Scope mouthwash were generally high for all microbial metrics. Similarly, the fecal comparability ICCs for RNAlater and FOBT cards were high, ranging from 0.63 (95% CI: 0.46, 0.75) for the relative abundance of Firmicutes to 0.93 (95% CI: 0.89, 0.96) for unweighted Unifrac. Comparability ICCs for OMNIgene ORAL and Scope mouthwash were lower than fecal ICCs, ranging from 0.55 (95% CI: 0.36, 0.70) for the Shannon index to 0.79 (95% CI: 0.69, 0.86) for Bray-Curtis. Overall, RNAlater, FOBT cards and Scope mouthwash were stable up to 4 days at room temperature. Samples collected using FOBT cards were generally comparable to RNAlater while the OMNIgene ORAL were less similar to Scope mouthwash.ConclusionsAs microbiome measures for feces samples collected using RNAlater, FOBT cards and oral samples collected using Scope mouthwash were stable over four days at room temperature, these would be most appropriate for microbial analyses in these populations. However, one collection method should be consistently since each method may induce some differences.

Project description:UnlabelledCommunity profiling of the oral microbiome requires the recovery of quality sequences in order to accurately describe microbial community structure and composition. Our objective was to assess the effects of specimen collection method, storage medium, and storage conditions on the relative abundance of taxa in saliva and plaque identified using 16S rRNA genes. We also assessed short-term changes in taxon composition and relative abundance and compared the salivary and dental plaque communities in children and adults. Over a 2-week period, four successive saliva and dental plaque specimens were collected from four adults with no dental decay (108 samples), and two successive specimens were collected from six children with four or more erupted teeth (48 samples). There were minimal differences in community composition at the phylum and operational taxonomic unit levels between dental plaque collection using a scaler and collection using a CytoSoft brush. Plaque samples stored in OMNIgene medium showed higher within-sample Shannon diversity, were compositionally different, and were more similar to each other than plaque stored in liquid dental transport medium. Saliva samples stored in OMNIgene recovered similar communities for at least a week following storage at room temperature. However, the microbial communities recovered from plaque and saliva stored in OMNIgene were significantly different in composition from their counterparts stored in liquid dental transport medium. Dental plaque communities collected from the same tooth type over four successive visits from the same adult did not significantly differ in structure or composition.ImportanceLarge-scale epidemiologic studies require collection over time and space, often with multiple teams collecting, storing, and processing data. Therefore, it is essential to understand how sensitive study results are to modest changes in collection and storage protocols that may occur with variation in personnel, resources available at a study site, and shipping requirements. The research presented in this paper measures the effects of multiple storage parameters and collection methodologies on the measured ecology of the oral microbiome from healthy adults and children. These results will potentially enable investigators to conduct oral microbiome studies at maximal efficiency by guiding informed administrative decisions pertaining to the necessary field or clinical work.

Project description:To our knowledge, fecal microbiota collection methods have not been evaluated in low- and middle-income countries. Therefore, we evaluated five different fecal sample collection methods for technical reproducibility, stability, and accuracy within the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh. Fifty participants from the HEALS provided fecal samples in the clinic which were aliquoted into no solution, 95% ethanol, RNAlater, postdevelopment fecal occult blood test (FOBT) cards, and fecal immunochemical test (FIT) tubes. Half of the aliquots were frozen immediately at -80°C (day 0) and the remaining samples were left at ambient temperature for 96 h and then frozen (day 4). Intraclass correlation coefficients (ICC) were calculated for the relative abundances of the top three phyla, for two alpha diversity measures, and for four beta diversity measures. The duplicate samples had relatively high ICCs for technical reproducibility at day 0 and day 4 (range, 0.79 to 0.99). The FOBT card and samples preserved in RNAlater and 95% ethanol had the highest ICCs for stability over 4 days. The FIT tube had lower stability measures overall. In comparison to the "gold standard" method using immediately frozen fecal samples with no solution, the ICCs for many of the microbial metrics were low, but the rank order appeared to be preserved as seen by the Spearman correlation. The FOBT cards, 95% ethanol, and RNAlater were effective fecal preservatives. These fecal collection methods are optimal for future cohort studies, particularly in low- and middle-income countries.IMPORTANCE The collection of fecal samples in prospective cohort studies is essential to provide the opportunity to study the effect of the human microbiota on numerous health conditions. However, these collection methods have not been adequately tested in low- and middle-income countries. We present estimates of technical reproducibility, stability at ambient temperature for 4 days, and accuracy comparing a "gold standard" for fecal samples in no solution, 95% ethanol, RNAlater, postdevelopment fecal occult blood test cards, and fecal immunochemical test tubes in a study conducted in Bangladesh. Fecal occult blood test cards and fecal samples stored in 95% ethanol or RNAlater adequately preserve fecal samples in this setting. Therefore, new studies in low- and middle-income countries should include collection of fecal samples using fecal occult blood test cards, 95% ethanol, or RNAlater for prospective cohort studies.

Dataset Information

Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

Background

Methods

Results

Conclusions

Impact

Publications

Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets