scholarly journals Comparative analysis of 16S rRNA gene and metagenome sequencing in pediatric gut microbiomes

2021 ◽  
Author(s):  
Danielle Peterson ◽  
Kevin S. Bonham ◽  
Sophie Rowland ◽  
Cassandra W. Pattanayak ◽  
Vanja Klepac-Ceraj ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Gut Microbiome ◽  
Age Groups ◽  
Alpha Diversity ◽  
Sequencing Depth ◽  
Human Infant ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomic Sequencing

AbstractThe colonization of the human gut microbiome begins at birth, and, over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon versus shotgun metagenomic sequencing techniques in 130 fecal samples; younger than 15, 15-30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

scholarly journals Comparative Analysis of 16S rRNA Gene and Metagenome Sequencing in Pediatric Gut Microbiomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Danielle Peterson ◽  
Kevin S. Bonham ◽  
Sophie Rowland ◽  
Cassandra W. Pattanayak ◽  
Vanja Klepac-Ceraj ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Gut Microbiome ◽  
Age Groups ◽  
Alpha Diversity ◽  
Sequencing Depth ◽  
Human Infant ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomic Sequencing

The colonization of the human gut microbiome begins at birth, and over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon vs. shotgun metagenomic sequencing techniques in 338 fecal samples; younger than 15, 15–30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

scholarly journals Yoghurt Consumption is Associated With Transient Changes in the Composition of the Human Gut Microbiome

2020 ◽  
Author(s):  
Caroline Ivanne Le Roy ◽  
Alexander Kurilshikov ◽  
Emily Leeming ◽  
Alessia Visconti ◽  
Ruth Bowyer ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Visceral Fat ◽  
Gut Microbiome ◽  
Body Weight Gain ◽  
Healthy Eating Index ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Shotgun Metagenomic Sequencing

Abstract Background: Yoghurt contains live bacteria that could contribute via modulation of the gut microbiota to its reported beneficial effects such as reduced body weight gain and lower incidence of type 2 diabetes. To date, the association between yoghurt consumption and the composition of the gut microbiota is underexplored. Here we used clinical variables, metabolomics, 16S rRNA and shotgun metagenomic sequencing data collected on over 1000 predominantly female UK twins to define the link between the gut microbiota and yoghurt-associated health benefits. Results: According to food frequency questionnaires (FFQ), 73% of subjects consumed yoghurt. Consumers presented a healthier diet pattern (healthy eating index: beta = 2.17±0.34; P = 2.72x10-10) and improved metabolic health characterised by reduced visceral fat (beta = -28.18±11.71 g; P = 0.01). According to 16S rRNA gene analyses and whole shotgun metagenomic sequencing approach consistent taxonomic variations were observed with yoghurt consumption. More specifically, we identified higher abundance of species used as yoghurt starters Streptococcus thermophilus (beta = 0.41±0.051; P = 6.14x10-12) and sometimes added Bifidobacterium animalis subsp. lactis (beta = 0.30±0.052; P = 1.49x10-8) in the gut of yoghurt consumers. Replication in 1103 volunteers from the LifeLines-DEEP cohort confirmed the increase of S. thermophilus among yoghurt consumers. Using food records collected the day prior to faecal sampling we showed that increase in these two yoghurt bacteria could be transient. Metabolomics analysis revealed that B. animalis subsp. lactis was associated with 13 faecal metabolites including a 3-hydroxyoctanoic acid, known to be involved in the regulation of gut inflammation.Conclusions: Yoghurt consumption is associated with reduced visceral fat mass and changes in gut microbiome including transient increase of yoghurt-contained species (i.e. S. thermophilus and B. lactis).

scholarly journals Yoghurt consumption is associated with transient changes in the composition of the human gut microbiome

2020 ◽  
Author(s):  
Caroline Ivanne Le Roy ◽  
Alexander Kurilshikov ◽  
Emily Leeming ◽  
Alessia Visconti ◽  
Ruth Bowyer ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Visceral Fat ◽  
Gut Microbiome ◽  
Body Weight Gain ◽  
Healthy Eating Index ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Shotgun Metagenomic Sequencing

Abstract Background: Yoghurt contains live bacteria that could contribute via modulation of the gut microbiota to its reported beneficial effects such as reduced body weight gain and lower incidence of type 2 diabetes. To date, the association between yoghurt consumption and the composition of the gut microbiota is underexplored. Here we used clinical variables, metabolomics, 16S rRNA and shotgun metagenomic sequencing data collected on over 1000 predominantly female UK twins to define the link between the gut microbiota and yoghurt-associated health benefits. Results: According to food frequency questionnaires (FFQ), 73% of subjects consumed yoghurt. Consumers presented a healthier diet pattern (healthy eating index: beta = 2.17±0.34; P = 2.72x10 -10 ) and improved metabolic health characterised by reduced visceral fat (beta = -28.18±11.71 g; P = 0.01). According to 16S rRNA gene analyses and whole shotgun metagenomic sequencing approach consistent taxonomic variations were observed with yoghurt consumption. More specifically, we identified higher abundance of species used as yoghurt starters Streptococcus thermophilus (beta = 0.41±0.051; P = 6.14x10 -12 ) and sometimes added Bifidobacterium animalis subsp. lactis (beta = 0.30±0.052; P = 1.49x10 -8 ) in the gut of yoghurt consumers. Replication in 1103 volunteers from the LL-DEEP cohort confirmed the increase of S. thermophilus among yoghurt consumers. Using food records collected the day prior to faecal sampling we showed that increase in these two yoghurt bacteria could be transient. Metabolomics analysis revealed that B. animalis subsp. lactis was associated with 13 faecal metabolites including a 3-hydroxyoctanoic acid, known to be involved in the regulation of gut inflammation. Conclusions: Yoghurt consumption is associated with reduced visceral fat mass and changes in gut microbiome including transient increase of yoghurt-contained species ( i.e. S. thermophilus and B. lactis ).

scholarly journals A unique and reliable fecal DNA extraction method for 16S rRNA gene and shotgun metagenomic sequencing in the analysis of the human gut microbiome

2020 ◽  
Author(s):  
Céline Elie ◽  
Magali Perret ◽  
Karen Louis ◽  
Asmaà Fritah-Lafont ◽  
Philippe Leissner ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Gut Microbiome ◽  
High Throughput Sequencing ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Human Gut ◽  
Human Gut Microbiome ◽  
Shotgun Metagenomic Sequencing

Abstract Background: The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to select a unique and best performing DNA extraction protocol for both metagenomic sequencing methods. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) in order to facilitate DNA extraction. Stool samples from nine healthy volunteers and nine patients with a Clostridium difficile infection were extracted with all protocols and sequenced with both metagenomic methods. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA, alpha-diversity, accuracy using a mock community of known composition and repeatability across technical replicates.Results: Independently of the sequencing methods used, SPD significantly improved efficiency of the four tested protocols compared with their commercial version, in terms of extracted DNA quality, accuracy of the predicted composition of the microbiota (notably for Gram-positive bacteria), sample alpha-diversity, and experimental repeatability. The best overall performance was obtained for the S-DQ protocol, SPD combined to the DNeasy PowerLyser PowerSoil protocol from QIAGEN.Conclusion: Based on this evaluation, we recommend to use the S-DQ protocol, to obtain standardized and high quality extracted DNA in the human gut microbiome studies.

scholarly journals Weaning age and its effect on the development of the swine gut microbiome and resistome

2021 ◽  
Author(s):  
Devin B Holman ◽  
Katherine E Gzyl ◽  
Kathy T Mou ◽  
Heather K Allen
Keyword(s):  
Relative Abundance ◽  
Gut Microbiome ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Carbohydrate Active Enzymes ◽  
Fecal Microbiome ◽  
Shotgun Metagenomic Sequencing ◽  
Swine Operations ◽  
Weaning Age

Piglets are often weaned between 19 and 22 d of age in North America although in some swine operations this may occur at 14 d or less. Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow's milk to a plant-based diet. The effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. In this study, pigs were weaned at either 14, 21, or 28 d of age and fecal samples collected 21 times from d 4 (neonatal) through to marketing at d 140. The fecal microbiome was characterized using 16S rRNA gene and shotgun metagenomic sequencing. The fecal microbiome of all piglets shifted significantly three to seven days post-weaning with an increase in microbial diversity. Several Prevotella spp. increased in relative abundance immediately after weaning as did butyrate-producing species such as Butyricicoccus porcorum, Faecalibacterium prausnitzii, and Megasphaera elsdenii. Within 7 days of weaning, the gut microbiome of pigs weaned at 21 and 28 days of age resembled that of pigs weaned at 14 d. Resistance genes to most antimicrobial classes decreased in relative abundance post-weaning with the exception of those conferring resistance to tetracyclines and macrolides-lincosamides-streptogramin B. The relative abundance of microbial carbohydrate-active enzymes (CAZymes) changed significantly in the post-weaning period with an enrichment of CAZymes involved in degradation of plant-derived polysaccharides. These results demonstrate that pigs tend to have a more similar microbiome as they age and that weaning age has only a temporary effect on the gut microbiome.

scholarly journals Identification of the relationship between the gut microbiome and feed efficiency in a commercial pig cohort

2021 ◽  
Author(s):  
Hui Jiang ◽  
Shaoming Fang ◽  
Hui Yang ◽  
Congying Chen
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Gut Microbiome ◽  
Feed Efficiency ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
The Relationship

Abstract Feed efficiency is an economically important trait in pig production. Gut microbiota plays an important role in energy harvest, nutrient metabolism and fermentation of dietary indigestible components. Whether and which gut microbes affect feed efficiency in pigs are largely unknown. Here, a total of 208 healthy Duroc pigs were used as experimental materials. Feces and serum samples were collected at the age of 140d. We first performed 16S rRNA gene and metagenomic sequencing analysis to investigate the relationship between the gut microbiome and porcine residual feed intake (RFI). 16S rRNA gene sequencing analysis detected 21 OTUs showing the tendency to correlation with the RFI (P < 0.01). Metagenomic sequencing further identified that the members of Clostridiales, e.g. Ruminococcus flavefaoiens, Lachnospiraceae bacterium 28-4 and Lachnospiraceae phytofermentans, were enriched in pigs with low RFI (high feed efficiency), while 11 bacterial species including five Prevotella spp., especially, the Prevotella copri, had higher abundance in pigs with high RFI. Functional capacity analysis suggested that the gut microbiome of low RFI pigs had high abundance of the pathways related to amino acid metabolism and biosynthesis, but low abundance of the pathways associated with monosaccharide metabolism and lipopolysaccharide biosynthesis. Serum metabolome and fecal short chain fatty acids (SCFAs) were determined by UPLC-QTOF/MS and gas chromatograph, respectively. Propionic acid in feces and the serum metabolites related to amino acid metabolism were negatively correlated with the RFI. The results from this study may provide potential gut microbial biomarkers that could be used for improving feed efficiency in pig production industry.

scholarly journals Dead or Alive? Molecular life-dead distinction in human stool samples reveals significantly different composition of the microbial community

2018 ◽  
Author(s):  
Alexandra Perras ◽  
Kaisa Koskinen ◽  
Maximilian Mora ◽  
Michael Beck ◽  
Lisa Wink ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Human Health ◽  
Gene Targeting ◽  
Gut Microbiome ◽  
Rrna Gene ◽  
Stool Samples ◽  
Human Stool

AbstractThe gut microbiome is strongly interwoven with human health. Conventional gut microbiome analysis generally involves 16S rRNA gene targeting next generation sequencing (NGS) of stool microbial communities, and correlation of results with clinical parameters. However, some microorganisms may not be alive at the time of sampling, and thus their impact on the human health is potentially less significant. As conventional NGS methods do not differentiate between viable and dead microbial components, retrieved results provide only limited information.Propidium monoazide (PMA) is frequently used in food safety monitoring and other disciplines to discriminate living from dead cells. PMA binds to free DNA and masks it for subsequent procedures. In this article we show the impact of PMA on the results of 16S rRNA gene-targeting NGS from human stool samples and validate the optimal applicable concentration to achieve a reliable detection of the living microbial communities.Fresh stool samples were treated with a concentration series of zero to 300 μM PMA, and were subsequently subjected to amplicon-based NGS. The results indicate that a substantial proportion of the human microbial community is not intact at the time of sampling. PMA treatment significantly reduced the diversity and richness of the sample depending on the concentration and impacted the relative abundance of certain important microorganisms (e.g. Akkermansia, Bacteroides). Overall, we found that a concentration of 100 μM PMA was sufficient to quench signals from disrupted microbial cells.The optimized protocol proposed here can be easily implemented in classical microbiome analyses, and helps to retrieve an improved and less blurry picture of the microbial community composition by excluding signals from background DNA.

scholarly journals Deep Investigating the Changes of Gut Microbiome and Its Correlation With the Shifts of Host Serum Metabolome Around Parturition in Sows

2021 ◽  
Vol 12 ◽  
Author(s):  
Hao Fu ◽  
Maozhang He ◽  
Jinyuan Wu ◽  
Yunyan Zhou ◽  
Shanlin Ke ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Gut Microbiome ◽  
Bacterial Species ◽  
Late Pregnancy ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Functional Capacities

Parturition is a crucial event in the sow reproduction cycle, which accompanies by a series of physiological changes, including sex hormones, metabolism, and immunity. More and more studies have indicated the changes of the gut microbiota from pregnancy to parturition. However, what bacterial species and functional capacities of the gut microbiome are changed around parturition has been largely unknown, and the correlations between the changes of gut bacterial species and host metabolome were also uncovered. In this study, by combining 16S rRNA gene and shotgun metagenomic sequencing data, and the profiles of serum metabolome and fecal short-chain fatty acids (SCFAs), we investigated the changes of gut microbiome, serum metabolite features and fecal SCFAs from late pregnancy (LP) to postpartum (PO) stage. We found the significant changes of gut microbiota from LP to PO stage in both 16S rRNA gene sequencing and metagenomic sequencing analyses. The bacterial species from Lactobacillus, Streptococcus, and Clostridium were enriched at the LP stage, while the species from Bacteroides, Escherichia, and Campylobacter had higher abundances at the PO stage. Functional capacities of the gut microbiome were also significantly changed and associated with the shifts of gut bacteria. Untargeted metabolomic analyses revealed that the metabolite features related to taurine and hypotaurine metabolism, and arginine biosynthesis and metabolism were enriched at the LP stage, and positively associated with those bacterial species enriched at the LP stage, while the metabolite features associated with vitamin B6 and glycerophospholipid metabolism had higher abundances at the PO stage and were positively correlated with the bacteria enriched at the PO stage. Six kinds of SCFAs were measured in feces samples and showed higher concentrations at the LP stage. These results suggested that the changes of gut microbiome from LP to PO stage lead to the shifts of host lipid, amino acids and vitamin metabolism and SCFA production. The results from this study provided new insights for the changes of sow gut microbiome and host metabolism around parturition, and gave new knowledge for guiding the feeding and maternal care of sows from late pregnancy to lactation in the pig industry.

scholarly journals Diet induces reproducible alterations in the mouse and human gut microbiome

2019 ◽  
Author(s):  
Jordan E. Bisanz ◽  
Vaibhav Upadhyay ◽  
Jessie A. Turnbaugh ◽  
Kimberly Ly ◽  
Peter J. Turnbaugh
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Meta Analysis ◽  
Alpha Diversity ◽  
Operational Taxonomic Unit ◽  
Molecular Testing ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Dietary Interventions

SummaryThe degree to which diet reproducibly alters the human and mouse gut microbiota remains unclear. Here, we focus on the consumption of a high-fat diet (HFD), one of the most frequently studied dietary interventions in mice. We employed a subject-level meta-analysis framework for unbiased collection and analysis of publicly available 16S rRNA gene and metagenomic sequencing data from studies examining HFD in rodent models. In total, we re-analyzed 27 studies, 1101 samples, and 106 million reads mapping to 16S rRNA gene sequences. We report reproducible changes in gut microbial community structure both within and between studies, including a significant increase in the Firmicutes phylum and decrease in the Bacteroidetes phylum; however, reduced alpha diversity is not a consistent feature of HFD. Finer taxonomic analysis revealed that the strongest signal of HFD on microbiota species composition is Lactococcus spp., which we demonstrate is a common dietary contaminant through the molecular testing of dietary ingredients, culturing, microscopy, and germ-free mouse experiments. After in silico removal of Lactococcus spp., we employed machine learning to define a unique operational taxonomic unit (OTU)-based signature capable of predicting the dietary intake of mice and demonstrate that phylogenetic and gene-family transformations of this model are capable of accurately predicting human samples in controlled feeding settings (area under the receiver operator curve = 0.75 and 0.88 respectively). Together, these results demonstrate the utility of microbiome meta-analyses in identifying robust bacterial signals for mechanistic studies and creates a framework for the routine meta-analysis of microbiome studies in preclinical models.

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