scholarly journals Comparison of Methods To Collect Fecal Samples for Microbiome Studies Using Whole-Genome Shotgun Metagenomic Sequencing

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Doratha A. Byrd ◽  
Rashmi Sinha ◽  
Kristi L. Hoffman ◽  
Jun Chen ◽  
Xing Hua ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Fecal Sample ◽  
Gold Standard ◽  
Beta Diversity ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Sample Collection ◽  
Fecal Samples ◽  
Shotgun Metagenomic Sequencing ◽  
Collection Methods

ABSTRACT Few previous studies have assessed stability and “gold-standard” concordance of fecal sample collection methods for whole-genome shotgun metagenomic sequencing (WGSS), an increasingly popular method for studying the gut microbiome. We used WGSS data to investigate ambient temperature stability and putative gold-standard concordance of microbial profiles in fecal samples collected and stored using fecal occult blood test (FOBT) cards, fecal immunochemical test (FIT) tubes, 95% ethanol, or RNAlater. Among 15 Mayo Clinic employees, for each collection method, we calculated intraclass correlation coefficients (ICCs) to estimate stability of fecal microbial profiles after storage for 4 days at ambient temperature and concordance with immediately frozen, no-solution samples (i.e., the putative gold standard). ICCs were estimated for multiple metrics, including relative abundances of select phyla, species, KEGG k-genes (representing any coding sequence that had >70% identity and >70% query coverage with respect to a known KEGG ortholog), KEGG modules, and KEGG pathways; species and k-gene alpha diversity; and Bray-Curtis and Jaccard species beta diversity. ICCs for microbial profile stability were excellent (≥90%) for fecal samples collected via most of the collection methods, except those preserved in 95% ethanol. Concordance with the immediately frozen, no-solution samples varied for all collection methods, but the number of observed species and the beta diversity metrics tended to have higher concordance than other metrics. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT cards, FIT tubes, and RNAlater are acceptable choices for fecal sample collection methods in future WGSS studies. IMPORTANCE A major direction for future microbiome research is implementation of fecal sample collections in large-scale, prospective epidemiologic studies. Studying microbiome-disease associations likely requires microbial data to be pooled from multiple studies. Our findings suggest collection methods that are most optimal to be used standardly across future WGSS microbiome studies.

scholarly journals Genomic evolution of antimicrobial resistance in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pimlapas Leekitcharoenphon ◽  
Markus Hans Kristofer Johansson ◽  
Patrick Munk ◽  
Burkhard Malorny ◽  
Magdalena Skarżyńska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Genes ◽  
Mobile Genetic Elements ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
E Coli ◽  
Genetic Elements

AbstractThe emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

scholarly journals Fecal Sample Collection Methods and Time of Day Impact Microbiome Composition and Short Chain Fatty Acid Concentrations

2021 ◽  
Author(s):  
Jacquelyn Jones ◽  
Stacey Reinke ◽  
Alishum Ali ◽  
Debra Palmer ◽  
Claus T. Christophersen
Keyword(s):  
Fatty Acid ◽  
Fecal Sample ◽  
Short Chain Fatty Acid ◽  
Time Of Day ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Its2 Region ◽  
Rrna Gene ◽  
Sample Collection ◽  
Collection Methods

Abstract Associations between the human gut microbiome and health outcomes continues to be of great interest, although fecal sample collection methods which impact microbiome studies are sometimes neglected. Here, we expand on previous work in sample optimization, to promote high quality microbiome data. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. We demonstrated that spot sampling of stool results in variable detection of some microbial members, and inconsistent levels of SCFA; therefore, sample homogenization prior to subsequent analysis or subsampling is recommended. We also identify a trend in microbial and metabolite composition that shifts over two consecutive stool collections less than 25h apart. Lastly, we show significant differences in bacterial composition that result from collecting stool samples in OMNIgeneGUT tube (DNA Genotec) or Stool Nucleic Acid Collection and Preservation Tube (NORGEN) compared to immediate freezing. To assist microbiome investigators plan their fecal sample collection and storage procedures for multiple analyses, we recommend participants to collect the first full bowel movement of the day and freeze the sample immediately after collection.

scholarly journals Metagenome-Assembled Genomes Isolated from a Human Fecal Diarrhea Sample

2021 ◽  
Vol 10 (19) ◽  
Author(s):  
Tshepiso Pleasure Ateba ◽  
Kazeem Adekunle Alayande ◽  
Ngoma Lubanza ◽  
Mulunda Mwanza
Keyword(s):  
Infectious Disease ◽  
Microbial Community ◽  
Fecal Sample ◽  
Metagenomic Sequencing ◽  
Affected Child ◽  
Shotgun Metagenomic Sequencing

ABSTRACT Diarrheal infection is the second leading infectious disease that is killing children under the age of 5 years. This study investigates the microbial community within a fecal sample from a diarrhea-affected child through shotgun metagenomic sequencing.

scholarly journals The maintenance of microbial community in human fecal samples by a cost effective preservation buffer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chongming Wu ◽  
Tianda Chen ◽  
Wenyi Xu ◽  
Tingting Zhang ◽  
Yuwei Pei ◽  
...  
Keyword(s):  
Storage Temperature ◽  
Cost Effective ◽  
Human Saliva ◽  
Fecal Microbiota ◽  
Microbial Consortia ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
Preservation Methods ◽  
Shotgun Metagenomic Sequencing ◽  
Cost Efficient

AbstractIn the burgeoning microbiome field, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. The proper preservation of samples before being processed is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4 h, independent of the storage temperature. We also verified a previous reported self-made preservation buffer (PB buffer) could not only preserve fecal microbiota at room temperature up to 4 weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize samples if neither freezing facilities nor liquid nitrogen is available.

scholarly journals Fecal sample collection methods and time of day impact microbiome composition and short chain fatty acid concentrations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jacquelyn Jones ◽  
Stacey N Reinke ◽  
Alishum Ali ◽  
Debra J Palmer ◽  
Claus T. Christophersen
Keyword(s):  
Fatty Acid ◽  
Fecal Sample ◽  
Short Chain Fatty Acid ◽  
Time Of Day ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Its2 Region ◽  
Rrna Gene ◽  
Sample Collection ◽  
Collection Methods

AbstractAssociations between the human gut microbiome and health outcomes continues to be of great interest, although fecal sample collection methods which impact microbiome studies are sometimes neglected. Here, we expand on previous work in sample optimization, to promote high quality microbiome data. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. We demonstrated that spot sampling of stool results in variable detection of some microbial members, and inconsistent levels of SCFA; therefore, sample homogenization prior to subsequent analysis or subsampling is recommended. We also identify a trend in microbial and metabolite composition that shifts over two consecutive stool collections less than 25 h apart. Lastly, we show significant differences in bacterial composition that result from collecting stool samples in OMNIgene·Gut tube (DNA Genotec) or Stool Nucleic Acid Collection and Preservation Tube (NORGEN) compared to immediate freezing. To assist with planning fecal sample collection and storage procedures for microbiome investigations with multiple analyses, we recommend participants to collect the first full bowel movement of the day and freeze the sample immediately after collection.

scholarly journals Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 819
Author(s):  
Michelle Martin de Bustamante ◽  
Caryn Plummer ◽  
Jennifer MacNicol ◽  
Diego Gomez
Keyword(s):  
Ambient Temperature ◽  
Room Temperature ◽  
High Throughput Sequencing ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Sample Storage ◽  
Sample Collection ◽  
Microbial Composition ◽  
Fecal Samples ◽  
Community Membership

Sample storage conditions are an important factor in fecal microbiota analyses in general. The objective of this study was to investigate the effect of sample storage at room temperature on the equine fecal microbiota composition. Fecal samples were collected from 11 healthy horses. Each sample was divided into 7 sealed aliquots. One aliquot was immediately frozen at −80 °C; the remaining aliquots were stored at room temperature (21 to 22 °C) with one transferred to the freezer at each of the following time points: 6, 12, 24, 48, 72 and 96 h. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. Fibrobacteraceae (Fibrobacter) and Ruminococcaceae (Ruminococcus) were enriched in samples from 0 h and 6 h, whereas taxa from the families Bacillaceae, Planococcaceae, Enterobacteriaceae and Moraxellaceae were enriched in samples stored at room temperature for 24 h or greater. Samples frozen within the first 12 h after collection shared similar community membership. The community structure was similar for samples collected at 0 h and 6 h, but it was significantly different between samples frozen at 0 h and 12 h or greater. In conclusion, storage of equine fecal samples at ambient temperature for up to 6 h before freezing following sample collection had minimal effect on the microbial composition. Longer-term storage at ambient temperature resulted in alterations in alpha-diversity, community membership and structure and the enrichment of different taxa when compared to fecal samples immediately frozen at −80 °C.

scholarly journals Investigation of the impact of stool collection methods on the metabolomics analysis/profiles of infant fecal samples

2021 ◽  
Author(s):  
Chiara-Maria Homann ◽  
Sara Dizzell ◽  
Sandi Azab ◽  
Eileen K Hutton ◽  
Katherine M Morrison ◽  
...  
Keyword(s):  
Stool Sample ◽  
Short Chain Fatty Acids ◽  
Sample Collection ◽  
Fecal Samples ◽  
Wide Range ◽  
Metabolite Profiles ◽  
Comprehensive Metabolomics ◽  
The Impact ◽  
Collection Methods ◽  
Metabolomics Analysis

Metabolomic studies are important to understand microbial metabolism and interaction between the host and the gut microbiome. Although there have been efforts to standardize sample processing in metabolomic studies, infant samples are mostly disregarded. In birth cohort studies, the use of diaper liners is prevalent and its impact on fecal metabolic profile remains untested. In this study, we compared metabolite profiles of fecal samples collected as solid stool and those collected from stool saturated liner. One infant's stool sample was collected in triplicate for solid stool and stool saturated liner. Comprehensive metabolomics analysis of the fecal samples was performed using NMR, UPLC and DI-MS. The total number, identities and concentrations of the metabolites were determined and compared between stool sample collection methods (stool vs. liner). The number and identity of metabolites did not differ between collection methods for NMR and DI-MS when excluding metabolites with a coefficient of variation (CV) > 40%. NMR analysis demonstrated lowest bias between collection methods, and lowest technical precision between triplicates of the same method followed by DI-MS then UPLC. Concentrations of many metabolites from stool and stool saturated liner differed significantly as revealed by Bland-Altman plots and t-tests. Overall, a mean bias of 10.2% in the Bland-Altman analysis was acceptable for some metabolites confirming mutual agreement but not for others with a wide range of bias (-97-117%). Consequently, stool and stool-saturated liner could be used interchangeably only for some select metabolite classes e.g. amino acids. Differences between the metabolomic profiles of solid stool samples and stool saturated liner samples for some important molecules e.g., ethanol, fumarate, short chain fatty acids and bile acids, indicate the need for standardization in stool collection method for metabolomic studies performed in infants.

The Maintenance of Microbial Community in Human Fecal Samples by a Self-made Cost-effective Preservation Buffer

2021 ◽  
Author(s):  
Chongming Wu ◽  
Tianda Chen ◽  
Wenyi Xu ◽  
Tingting Zhang ◽  
Yuwei Pei ◽  
...  
Keyword(s):  
Storage Temperature ◽  
Cost Effective ◽  
Human Saliva ◽  
Fecal Microbiota ◽  
Microbial Consortia ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
Preservation Methods ◽  
Shotgun Metagenomic Sequencing ◽  
Cost Efficient

Abstract In the burgeoning microbiome filed, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. Maintaining sequencing samples is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that in a short-term storage, microbial consortia in human fecal samples were substantially maintained, independent of the storage temperature. We also verified a self-made preservation buffer could not only maintain fecal microbiota at ambient temperature up to several weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize sequencing samples if neither freezing facilities nor liquid nitrogen is available.

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