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.

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 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 Assessment of fecal DNA extraction protocols for metagenomic studies

GigaScience ◽  
2020 ◽  
Vol 9 (7) ◽  
Author(s):  
Fangming Yang ◽  
Jihua Sun ◽  
Huainian Luo ◽  
Huahui Ren ◽  
Hongcheng Zhou ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Cost Effective ◽  
Metagenomic Sequencing ◽  
Human Gut ◽  
Shotgun Metagenomics ◽  
Fecal Samples ◽  
Bead Size ◽  
Standardized Protocol ◽  
Fungal Dna ◽  
Fungal Dna Extraction

Abstract Background Shotgun metagenomic sequencing has improved our understanding of the human gut microbiota. Various DNA extraction methods have been compared to find protocols that robustly and most accurately reflect the original microbial community structures. However, these recommendations can be further refined by considering the time and cost demands in dealing with samples from very large human cohorts. Additionally, fungal DNA extraction performance has so far been little investigated. Results We compared 6 DNA extraction protocols, MagPure Fast Stool DNA KF Kit B, Macherey Nagel™ NucleoSpin™®Soil kit, Zymo Research Quick-DNA™ Fecal/Soil Microbe kit, MOBIO DNeasy PowerSoil kit, the manual non-commercial protocol MetaHIT, and the recently published protocol Q using 1 microbial mock community (MMC) (containing 8 bacterial and 2 fungal strains) and fecal samples. All samples were manually extracted and subjected to shotgun metagenomics sequencing. Extracting DNA revealed high reproducibility within all 6 protocols, but microbial extraction efficiencies varied. The MMC results demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. In human fecal samples, the MagPure bacterial extraction performed as well as the standardized protocol Q but was faster and more cost-effective. Extraction using the PowerSoil protocol resulted in a significantly higher ratio of gram-negative to gram-positive bacteria than other protocols, which might contribute to reported gut microbial differences between healthy adults. Conclusions We emphasize the importance of bead size selection for bacterial and fungal DNA extraction. More importantly, the performance of the novel protocol MP matched that of the recommended standardized protocol Q but consumed less time, was more cost-effective, and is recommended for further large-scale human gut metagenomic studies.

scholarly journals Fecal microbiota transplantation brings about bacterial strain displacement in patients with inflammatory bowel diseases

2018 ◽  
Author(s):  
Manli Zou ◽  
Zhuye Jie ◽  
Bota Cui ◽  
Honggang Wang ◽  
Qiang Feng ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Fecal Microbiota Transplantation ◽  
Bacterial Colonization ◽  
Fecal Microbiota ◽  
Classification Model ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
Inflammatory Bowel

ABSTRACTFecal microbiota transplantation (FMT), which is thought to have the potential to correct dysbiosis of gut microbiota, has recently been used to treat inflammatory bowel disease (IBD). To elucidate the extent and principles of microbiota engraftment in IBD patients after FMT treatment, we conducted an interventional prospective cohort study. The cohort included two categories of patients: (1) patients with moderate to severe Crohn’s disease (CD) (Harvey-Bradshaw Index ≥ 7, n = 11, and (2) patients with ulcerative colitis (UC) (Montreal classification, S2 and S3, n = 4). All patients were treated with a single FMT (via mid-gut, from healthy donors) and follow-up visits were performed at baseline, 3 days, one week, and one month after FMT (missing time points included). At each follow-up time point, fecal samples of the participants were collected along with their clinical metadata. For comparative analysis, 10 fecal samples from 10 healthy people were included to represent the diversity level of normal gut microbiota. Additionally, the metagenomic data of 25 fecal samples from 5 individuals with metabolic syndrome who underwent autologous FMT treatment were downloaded from a previous published paper to represent natural microbiota shifts during FMT. All fecal samples underwent shotgun metagenomic sequencing.We found that 3 days after FMT, 11 out of 15 recipients were in remission (3 out of 4 UC recipients; 8 out of 11 CD recipients). Generally, bacterial colonization was observed to be lower in CD recipients than in UC recipients at both species and strain levels. Furthermore, across species, different strains displayed disease-specific displacement advantages under two-disease status. Finally, most post-FMT species (> 80%) could be properly predicted (AUC > 85%) using a random forest classification model, with the gut microbiota composition and clinical parameters of pre-FMT recipients acting as the most contributive factors for prediction accuracy.

scholarly journals Domestication Shapes the Community Structure and Functional Metagenomic Content of the Yak Fecal Microbiota

2021 ◽  
Vol 12 ◽  
Author(s):  
Haibo Fu ◽  
Liangzhi Zhang ◽  
Chao Fan ◽  
Chuanfa Liu ◽  
Wenjing Li ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Short Chain Fatty Acids ◽  
Fecal Microbiota ◽  
Glycoside Hydrolase Family ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomic Sequencing ◽  
Rdna Sequencing ◽  
Key Factor ◽  
Methane Metabolism ◽  
Hydrolase Family

Domestication is a key factor of genetic variation; however, the mechanism by which domestication alters gut microbiota is poorly understood. Here, to explore the variation in the structure, function, rapidly evolved genes (REGs), and enzyme profiles of cellulase and hemicellulose in fecal microbiota, we studied the fecal microbiota in wild, half-blood, and domestic yaks based on 16S rDNA sequencing, shotgun-metagenomic sequencing, and the measurement of short-chain-fatty-acids (SCFAs) concentration. Results indicated that wild and half-blood yaks harbored an increased abundance of the phylum Firmicutes and reduced abundance of the genus Akkermansia, which are both associated with efficient energy harvesting. The gut microbial diversity decreased in domestic yaks. The results of the shotgun-metagenomic sequencing showed that the wild yak harbored an increased abundance of microbial pathways that play crucial roles in digestion and growth of the host, whereas the domestic yak harbored an increased abundance of methane-metabolism-related pathways. Wild yaks had enriched amounts of REGs in energy and carbohydrate metabolism pathways, and possessed a significantly increased abundance of cellulases and endohemicellulases in the glycoside hydrolase family compared to domestic yaks. The concentrations of acetic, propionic, n-butyric, i-butyric, n-valeric, and i-valeric acid were highest in wild yaks. Our study displayed the domestic effect on the phenotype of composition, function in gut microbiota, and SCFAs associated with gut microbiota, which had a closely association with the growth performance of the livestock. These findings may enlighten the researchers to construct more links between economic characteristics and gut microbiota, and develop new commercial strains in livestock based on the biotechnology of gut microbiota.

scholarly journals Microbial Community Profiling of Human Saliva Using Shotgun Metagenomic Sequencing

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e97699 ◽  
Author(s):  
Nur A. Hasan ◽  
Brian A. Young ◽  
Angela T. Minard-Smith ◽  
Kelly Saeed ◽  
Huai Li ◽  
...  
Keyword(s):  
Microbial Community ◽  
Human Saliva ◽  
Metagenomic Sequencing ◽  
Microbial Community Profiling ◽  
Community Profiling ◽  
Shotgun Metagenomic Sequencing

scholarly journals Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer

2021 ◽  
Vol 41 ◽  
pp. 01001
Author(s):  
Takuji Yamada
Keyword(s):  
Colorectal Cancer ◽  
Gut Microbiota ◽  
Nature Medicine ◽  
Cancer Control ◽  
Fusobacterium Nucleatum ◽  
Fecal Microbiota ◽  
Metagenomic Sequencing ◽  
Diagnostic Significance ◽  
Shotgun Metagenomic Sequencing ◽  
Sporadic Crcs

Colorectal cancer (CRC) worldwide affects over a quarter of a million people each year. Most sporadic CRCs develop through formation of polypoid adenomas and are preceded by intramucosal carcinoma (Stage 0), which can progress into malignant forms. Detection of early cancers and their endoscopic removal are priorities for cancer control. Human gut microbiome has been associated to CRC development, and its comprehensive characterization is of a great importance to assess its potential as a diagnostic marker. We performed whole shotgun metagenomic sequencing and CE-TOFMS-based metabolomic studies on fecal samples collected from 616 participants undergoing colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. As a result, microbiome and metabolite shifts were apparent in cases of multiple polypoid adenomas (MP) and Stage 0, in addition to more advanced lesions (Stage I/II and Stage III/IV). Two distinct patterns of microbiome elevations were found (P<0.005). First, CRC-associated species including Fusobacterium nucleatum were elevated continuously from Stage 0 to more advanced stages. Second, Atopobium parvulum and Actinomyces odontolyticus, which co-occurred in Stage 0, were elevated only in MP and/or in Stage 0. Metabolome analyses showed elevation of branched-chain amino acids and phenylalanine in Stage 0 and bile acids including deoxycholate in MP and/or Stage 0. Metagenomic functional analyses showed amino acid metabolism and sulfide producing pathways were associated with CRCs. Our study indicates possible etiological and diagnostic significance of fecal microbiota and metabolite in the very early stages of CRC (Yachida et al., Nature Medicine 2019).

scholarly journals Evaluation of the effects of library preparation procedure and sample characteristics on the accuracy of metagenomic profiles

2021 ◽  
Author(s):  
Christopher Gaulke ◽  
Emily R Schmeltzer ◽  
Mark Dasenko ◽  
Brett M Tyler ◽  
Rebecca Vega Thurber ◽  
...  
Keyword(s):  
Microbial Community ◽  
Cost Effective ◽  
Low Complexity ◽  
Careful Consideration ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Library Preparation ◽  
Dna Amount ◽  
Preparation Methods ◽  
Shotgun Metagenomic Sequencing

Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities, or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera® Flex kit, perform when applied to metagenomic DNA. We measured each method's ability to produce metagenomic data that accurately represents the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral-associated, mouse-gut-associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method’s performance, indicating that careful consideration may be needed when selecting between methods, especially for low complexity communities. However, cost-effective preparation methods we assessed are generally comparable to the current gold standard Nextera® DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies.

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