Accessing Dietary Effects on the Rumen Microbiome: Different Sequencing Methods Tell Different Stories

The current study employed both amplicon and shotgun sequencing to examine and compare the rumen microbiome in Angus bulls fed with either a backgrounding diet (BCK) or finishing diet (HG), to assess if both methods produce comparable results. Rumen digesta samples from 16 bulls were subjected for microbial profiling. Distinctive microbial profiles were revealed by the two methods, indicating that choice of sequencing approach may be a critical facet in studies of the rumen microbiome. Shotgun-sequencing identified the presence of 303 bacterial genera and 171 archaeal species, several of which exhibited differential abundance. Amplicon-sequencing identified 48 bacterial genera, 4 archaeal species, and 9 protozoal species. Among them, 20 bacterial genera and 5 protozoal species were differentially abundant between the two diets. Overall, amplicon-sequencing showed a more drastic diet-derived effect on the ruminal microbial profile compared to shotgun-sequencing. While both methods detected dietary differences at various taxonomic levels, few consistent patterns were evident. Opposite results were seen for the phyla Firmicutes and Bacteroidetes, and the genus Selenomonas. This study showcases the importance of sequencing platform choice and suggests a need for integrative methods that allow robust comparisons of microbial data drawn from various omic approaches, allowing for comprehensive comparisons across studies.

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CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems

Microorganisms ◽

10.3390/microorganisms9040816 ◽

2021 ◽

Vol 9 (4) ◽

pp. 816

Author(s):

Matthew G. Links ◽

Tim J. Dumonceaux ◽

E. Luke McCarthy ◽

Sean M. Hemmingsen ◽

Edward Topp ◽

...

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Dna Sequences ◽

Pcr Amplification ◽

Shotgun Sequencing ◽

Natural Ecosystems ◽

Gene Markers ◽

Microbial Profile ◽

Microbial Ecosystems ◽

Pcr Targeting

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

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Reduced metagenome sequencing for strain-resolution taxonomic profiles

Microbiome ◽

10.1186/s40168-021-01019-8 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Lars Snipen ◽

Inga-Leena Angell ◽

Torbjørn Rognes ◽

Knut Rudi

Keyword(s):

Microbial Community ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Good Alternative ◽

Shotgun Sequencing ◽

Least Square ◽

Full Potential ◽

Shotgun Metagenomics ◽

Metagenome Sequencing ◽

Reference Genomes

Abstract Background Studies of shifts in microbial community composition has many applications. For studies at species or subspecies levels, the 16S amplicon sequencing lacks resolution and is often replaced by full shotgun sequencing. Due to higher costs, this restricts the number of samples sequenced. As an alternative to a full shotgun sequencing we have investigated the use of Reduced Metagenome Sequencing (RMS) to estimate the composition of a microbial community. This involves the use of double-digested restriction-associated DNA sequencing, which means only a smaller fraction of the genomes are sequenced. The read sets obtained by this approach have properties different from both amplicon and shotgun data, and analysis pipelines for both can either not be used at all or not explore the full potential of RMS data. Results We suggest a procedure for analyzing such data, based on fragment clustering and the use of a constrained ordinary least square de-convolution for estimating the relative abundance of all community members. Mock community datasets show the potential to clearly separate strains even when the 16S is 100% identical, and genome-wide differences is < 0.02, indicating RMS has a very high resolution. From a simulation study, we compare RMS to shotgun sequencing and show that we get improved abundance estimates when the community has many very closely related genomes. From a real dataset of infant guts, we show that RMS is capable of detecting a strain diversity gradient for Escherichia coli across time. Conclusion We find that RMS is a good alternative to either metabarcoding or shotgun sequencing when it comes to resolving microbial communities at the strain level. Like shotgun metagenomics, it requires a good database of reference genomes and is well suited for studies of the human gut or other communities where many reference genomes exist. A data analysis pipeline is offered, as an R package at https://github.com/larssnip/microRMS.

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Deep Illumina-Based Shotgun Sequencing Reveals Dietary Effects on the Structure and Function of the Fecal Microbiome of Growing Kittens

PLoS ONE ◽

10.1371/journal.pone.0101021 ◽

2014 ◽

Vol 9 (7) ◽

pp. e101021 ◽

Cited By ~ 23

Author(s):

Oliver Deusch ◽

Ciaran O’Flynn ◽

Alison Colyer ◽

Penelope Morris ◽

David Allaway ◽

...

Keyword(s):

Structure And Function ◽

Shotgun Sequencing ◽

Dietary Effects ◽

Fecal Microbiome ◽

And Function

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Combining whole genome shotgun sequencing and rDNA amplicon analyses to improve detection of microbe-microbe interaction networks in plant leaves

10.1101/823492 ◽

2019 ◽

Cited By ~ 2

Author(s):

Julian Regalado ◽

Derek S. Lundberg ◽

Oliver Deusch ◽

Sonja Kersten ◽

Talia Karasov ◽

...

Keyword(s):

16S Rdna ◽

High Throughput Sequencing ◽

Cost Effective ◽

Amplicon Sequencing ◽

Low Complexity ◽

Shotgun Sequencing ◽

Wild Plants ◽

Hybrid Strategy ◽

Domains Of Life ◽

Metagenome Sequencing

AbstractMicroorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, acquire nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of 16S rDNA and/or the internal transcribed spacer (ITS) of rDNA loci, but the decreasing cost of high-throughput sequencing has made shotgun metagenome sequencing increasingly accessible. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S rDNA and eukaryotic ITS1 amplicon data from 176 of these samples. The shotgun data were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. For shotgun and amplicon data, microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. We use the metagenome data, which captures the ratio of bacterial to plant DNA in leaves of wild plants, to scale the 16S rDNA amplicon data such that they reflect absolute bacterial abundance. We show that this cost-effective hybrid strategy overcomes compositionality problems in amplicon data and leads to fundamentally different conclusions about microbiome community assembly.

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Changes in the gut microbiota during and after commercial helium–oxygen saturation diving in China

Occupational and Environmental Medicine ◽

10.1136/oemed-2019-106031 ◽

2019 ◽

Vol 76 (11) ◽

pp. 801-807

Author(s):

Yuan Yuan ◽

Guosheng Zhao ◽

Hongwei Ji ◽

Bin Peng ◽

Zhiguo Huang ◽

...

Keyword(s):

Gut Microbiota ◽

Oxygen Saturation ◽

Amplicon Sequencing ◽

Obligate Anaerobe ◽

Sequencing Platform ◽

Β Diversity ◽

Faecal Samples ◽

Increasing Trend ◽

Hyperbaric Environment ◽

Illumina Sequencing Platform

ObjectivesThe influence of commercial helium–oxygen saturation diving on divers’ gut microbiotas was assessed to provide dietary suggestion.MethodsFaecal samples of 47 divers working offshore were collected before (T1), during (T2) and after (T3) saturation diving. Their living and excursion depths were 55–134 metres underwater with a saturation duration of 12–31 days and PaO2 of 38–65 kPa. The faecal samples were examined through 16S ribosomal DNA amplicon sequencing based on the Illumina sequencing platform to analyse changes in the bacteria composition in the divers’ guts.ResultsAlthough the α and β diversity of the gut microbiota did not change significantly, we found that living in a hyperbaric environment of helium–oxygen saturation decreased the abundance of the genus Bifidobacterium, an obligate anaerobe, from 2.43%±3.83% at T1 to 0.79%±1.23% at T2 and 0.59%±0.79% at T3. Additionally, the abundance of some short-chain fatty acid (SCFA)-producing bacteria, such as Fusicatenibacter, Faecalibacterium, rectale group and Anaerostipes, showed a decreased trend in the order of before, during and after diving. On the contrary, the abundance of species, such as Lactococcus garvieae, Actinomyces odontolyticus, Peptoclostridium difficile, Butyricimonas virosa, Streptococcus mutans, Porphyromonas asaccharolytica and A. graevenitzii, showed an increasing trend, but most of them were pathogens.ConclusionsOccupational exposure to high pressure in a helium–oxygen saturation environment decreased the abundance of Bifidobacterium and some SCFA-producing bacteria, and increased the risk of pathogenic bacterial infection. Supplementation of the diver diet with probiotics or prebiotics during saturation diving might prevent these undesirable changes.

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Meta-Analysis of Microbial Communities in Hot Springs: Recurrent Taxa and Complex Shaping Factors beyond pH and Temperature

Microorganisms ◽

10.3390/microorganisms8060906 ◽

2020 ◽

Vol 8 (6) ◽

pp. 906 ◽

Cited By ~ 2

Author(s):

Francisco L. Massello ◽

Chia Sing Chan ◽

Kok-Gan Chan ◽

Kian Mau Goh ◽

Edgardo Donati ◽

...

Keyword(s):

Microbial Communities ◽

Metabolic Profile ◽

Hot Springs ◽

Meta Analysis ◽

Extreme Environments ◽

Alpha Diversity ◽

Amplicon Sequencing ◽

Phylogenetic Distance ◽

Microbial Profile ◽

The World

The study of microbial communities from extreme environments is a fascinating topic. With every study, biologists and ecologists reveal interesting facts and questions that dispel the old belief that these are inhospitable environments. In this work, we assess the microbial diversity of three hot springs from Neuquén, Argentina, using high-throughput amplicon sequencing. We predicted a distinct metabolic profile in the acidic and the circumneutral samples, with the first ones being dominated by chemolithotrophs and the second ones by chemoheterotrophs. Then, we collected data of the microbial communities of hot springs around the world in an effort to comprehend the roles of pH and temperature as shaping factors. Interestingly, there was a covariation between both parameters and the phylogenetic distance between communities; however, neither of them could explain much of the microbial profile in an ordination model. Moreover, there was no correlation between alpha diversity and these parameters. Therefore, the microbial communities’ profile seemed to have complex shaping factors beyond pH and temperature. Lastly, we looked for taxa associated with different environmental conditions. Several such taxa were found. For example, Hydrogenobaculum was frequently present in acidic springs, as was the Sulfolobaceae family; on the other hand, Candidatus Hydrothermae phylum was strongly associated with circumneutral conditions. Interestingly, some singularities related to sites featuring certain taxa were also observed.

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Gut microbiota composition is associated with narcolepsy type 1

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000896 ◽

2020 ◽

Vol 7 (6) ◽

pp. e896

Author(s):

Alexandre Lecomte ◽

Lucie Barateau ◽

Pedro Pereira ◽

Lars Paulin ◽

Petri Auvinen ◽

...

Keyword(s):

Community Structure ◽

Gut Microbiota ◽

Bacterial Communities ◽

Beta Diversity ◽

Alpha Diversity ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Differential Abundance ◽

Alpha And Beta Diversity

ObjectiveTo test the hypothesis that narcolepsy type 1 (NT1) is related to the gut microbiota, we compared the microbiota bacterial communities of patients with NT1 and control subjects.MethodsThirty-five patients with NT1 (51.43% women, mean age 38.29 ± 19.98 years) and 41 controls (57.14% women, mean age 36.14 ± 12.68 years) were included. Stool samples were collected, and the fecal microbiota bacterial communities were compared between patients and controls using the well-standardized 16S rRNA gene amplicon sequencing approach. We studied alpha and beta diversity and differential abundance analysis between patients and controls, and between subgroups of patients with NT1.ResultsWe found no between-group differences for alpha diversity, but we discovered in NT1 a link with NT1 disease duration. We highlighted differences in the global bacterial community structure as assessed by beta diversity metrics even after adjustments for potential confounders as body mass index (BMI), often increased in NT1. Our results revealed differential abundance of several operational taxonomic units within Bacteroidetes, Bacteroides, and Flavonifractor between patients and controls, but not after adjusting for BMI.ConclusionWe provide evidence of gut microbial community structure alterations in NT1. However, further larger and longitudinal multiomics studies are required to replicate and elucidate the relationship between the gut microbiota, immunity dysregulation and NT1.

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Maternal influences on beef calf rumen microbiome in the first 4 wk of life

Translational Animal Science ◽

10.1093/tas/txy077 ◽

2018 ◽

Vol 2 (suppl_1) ◽

pp. S96-S96

Author(s):

S Powell ◽

H C Cunningham ◽

K J Austin ◽

K M Cammack

Keyword(s):

Treatment Group ◽

Microbial Diversity ◽

Mode Of Delivery ◽

Caesarian Section ◽

Vaginal Birth ◽

Differential Abundance ◽

Maternal Influences ◽

Treatment Groups ◽

Β Diversity ◽

Rumen Microbiome

Abstract The preruminant microbiome has the potential to set the stage for later life feed efficiency and is critical to proper development within the rumen. We hypothesized that the rumen microbiome is established at or near birth and is subject to maternal influences that can influence preruminant and postruminant microbial profiles. Our objective was to determine how mode of delivery and rearing affected the development of the rumen microbiome. Bred mature Charolais cows were randomly allocated to one of the three treatment groups: control (CON; n = 8), bottle reared (BOT; n = 8), and caesarian section (CSET; n = 8), where CON was vaginal birth and raised by their dam; BOT was vaginal birth, then removed 24-h post-parturition, and raised on commercial milk replacer; and CSET was born via caesarian section and raised by their respective dams. Calf rumen fluid was collected from calves at 1, 3, and 28 d of age via oral lavage and metagenomic shotgun sequencing was performed using the Illumina NextSeq 500 platform. Sequence data were analyzed utilizing Metataxa2 for taxonomic assignment followed by QIIME to determine α- and β-diversity differences. A total of 1,113 taxa had differential abundance when comparing day while 66 taxa had differential abundance across treatment groups. There were no differences across treatment group richness (P > 0.05), but day 28 was significantly more rich (P = 0.003) compared with days 1 and 3 with no difference between days 1 and 3 (P = 0.58). No differences in β-diversity were detected across treatment group with the exception of greater variance in the BOT and CSET compared with the CON (P = 0.048). Microbial profiles of day 1 are more similar to each other than day 3 or 28 (P = 0.03); day 3 is more similar to each other than day 1 or 28 (P = 0.03); and day 28 is more similar to each other than day 1 or 3 (P = 0.03). These data suggest that while treatment group did not have a large impact on microbial diversity, several specific taxa were affected by treatment group. Day affects the microbial diversity both within and among samples. Understanding how these profiles shift with age is critical to understanding key intervention periods for optimal alteration of the microbiome.

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PSV-16 Influence of the late gestation maternal rumen microbiome on the calf meconium and early rumen microbiome

Journal of Animal Science ◽

10.1093/jas/skaa054.290 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 164-164

Author(s):

Kelly Woodruff ◽

Gwendolynn Hummel ◽

Kathleen Austin ◽

Travis Smith ◽

Hannah Cunningham-Hollinger

Keyword(s):

Beta Diversity ◽

Rumen Fluid ◽

Amplicon Sequencing ◽

Late Gestation ◽

Sample Type ◽

Alpha And Beta Diversity ◽

Rumen Microbiome ◽

Beef Herd ◽

The University ◽

Microbial Dna

Abstract Understanding the development of the calf rumen microbiome is important in developing manipulation strategies to improve efficiency as the animal ages. We hypothesized that the cow maternal microbiome would influence the colonization of the calf rumen microbiome. Our objective was to relate the microbiomes of the cow rumen fluid (RFC) to the calf meconium (M) and calf rumen fluid (RFN) at twenty-eight days of age. Mature, multiparous Angus crossbred cows (n = 10) from the University of Wyoming beef herd were used in this study. Rumen fluid was collected from the cows prior to parturition. Immediately following parturition, meconium was collected from the calf and at 28 days post-parturition, rumen fluid was collected from the calves. Microbial DNA was isolated using a lysis buffer and mechanical bead-beating procedure and purified using the QIAamp DNA Stool Mini Kit (Qiagen). Amplicon sequencing of the 16S rRNA V4 region was completed on the MiSeq and analyzed with QIIME2. Both alpha and beta diversity were evaluated by sample type and day. Richness and evenness differed by sample type. The greatest richness and evenness was in RFC (q < 0.01) followed by RFN and M, which did not differ from each other (q ³ 0.5). Bray-Curtis and Jaccard beta diversity differed by each sample type (q < 0.01). These data indicate that the M and RFN do not differ in number and distribution of features, but the samples are compositionally different. Additionally, the RFC differed in both alpha and beta diversity from both calf samples. These profiles can be used to develop hypotheses for the pathway of colonization in the early gut yet still reflect the vast differences in the developmental stage between the cow rumen microbiome and the early calf gastrointestinal microbiome.

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