scholarly journals The Milk Microbiota of the Spanish Churra Sheep Breed: New Insights into the Complexity of the Milk Microbiome of Dairy Species

Animals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1463
Author(s):  
Cristina Esteban-Blanco ◽  
Beatriz Gutiérrez-Gil ◽  
Héctor Marina ◽  
Rocío Pelayo ◽  
Aroa Suárez-Vega ◽  
...  
Keyword(s):  
Animal Species ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Rrna Gene ◽  
Microbiota Composition ◽  
Complex Issue ◽  
The Core ◽  
Sheep Milk ◽  
The 16S Rrna Gene ◽  
Generation Sequencing

Milk from healthy animals has classically been considered a sterile fluid. With the development of massively parallel sequencing and its application to the study of the microbiome of different body fluids, milk microbiota has been documented in several animal species. In this study, the main objective of this work was to access bacterial profiles of healthy milk samples using the next-generation sequencing of amplicons from the 16S rRNA gene to characterise the milk microbiome of the Churra breed. A total of 212 samples were collected from two Churra dairy farms with a different management system. The core milk microbiota in Churra ewes includes lesser genera (only two taxa: Staphylococcus and Escherichia/Shigella) than studies reported in other dairy species or even in a previous study in Assaf sheep milk. We found that diversity values in the two flocks of Churra breed were lower than the diversity of the milk microbiota in Assaf. The non-metric multidimensional scaling (NMDS) ordination using Bray-Curtis distance separates samples based on their microbiota composition. The information reported here might be used to understand the complex issue of milk microbiota composition.

scholarly journals Influence of Traditional vs Alternative Dietary Carbohydrates Sources on the Large Intestinal Microbiota in Post-Weaning Piglets

Animals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 516 ◽  
Author(s):  
Tretola ◽  
Luciano ◽  
Ottoboni ◽  
Baldi ◽  
Pinotti
Keyword(s):  
Growth Performance ◽  
Rrna Gene ◽  
Microbiota Composition ◽  
Gut Health ◽  
Faecal Microbiota ◽  
Alternative Ingredients ◽  
Weaning Piglets ◽  
The 16S Rrna Gene ◽  
Generation Sequencing ◽  
Gut Microbiota Composition

In this study, common cereal grains were partially replaced by former foodstuffs products (FFPs) in post-weaning piglets’ diets, to investigate how these alternative ingredients influence the faecal microbiota in the post-weaning period. Twelve post-weaning piglets were housed for 16 days in individual pens and were then fed two diets: a standard wheat-barley-corn meal diet and a diet containing 30% FFPs, thus partially substituting conventional cereals. The growth performance was monitored and faecal microbiota was characterized by the next generation sequencing of the 16S rRNA gene. The results showed no detrimental effects on growth performance when FFPs were used. However, the FFP diet decreased the bacterial richness and evenness in the large intestine, while minor differences were observed in the taxa composition. The core microbiota composition was only slightly affected, and no differences between the two groups in the gut microbiota composition at the phylum level over time were observed. Thus, although these results should be interpreted with caution, as they are case-specific, FFPs can be potentially used as alternative carbohydrate sources in post-weaning piglets, but further investigations are necessary to clarify their impact on gut health when used for a longer period.

scholarly journals Comparison of Normalization Methods for Construction of Large, Multiplex Amplicon Pools for Next-Generation Sequencing

2010 ◽  
Vol 76 (12) ◽  
pp. 3863-3868 ◽  
Author(s):  
J. Kirk Harris ◽  
Jason W. Sahl ◽  
Todd A. Castoe ◽  
Brandie D. Wagner ◽  
David D. Pollock ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Massively Parallel Sequencing ◽  
Sequence Data ◽  
Cost Savings ◽  
Massively Parallel ◽  
Next Generation ◽  
Normalization Methods ◽  
The Cost ◽  
Generation Sequencing

ABSTRACT Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.

scholarly journals Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

2006 ◽  
Vol 189 (3) ◽  
pp. 818-832 ◽  
Author(s):  
K. K. Hill ◽  
T. J. Smith ◽  
C. H. Helma ◽  
L. O. Ticknor ◽  
B. T. Foley ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Common Property ◽  
Animal Species ◽  
Fragment Length Polymorphism ◽  
Aflp Analysis ◽  
Rrna Gene ◽  
Botulinum Neurotoxins ◽  
The Common ◽  
Multiple Strains ◽  
The 16S Rrna Gene

ABSTRACT Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

scholarly journals Amplification of the V5 – V8 region of the 16S rRNA gene effectively speciates medically important genital tract Lactobacillus species in the upper female genital tract

2019 ◽  
Author(s):  
Jessica L. O’Callaghan ◽  
Dana Willner ◽  
Melissa Buttini ◽  
Flavia Huygens ◽  
Elise S. Pelzer
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Genital Tract ◽  
Rrna Gene ◽  
Next Generation ◽  
Sequencing Technology ◽  
Next Generation Sequencing Technology ◽  
The 16S Rrna Gene ◽  
Generation Sequencing

ABSTRACTBackgroundThe endometrial cavity is an upper genital tract site largely heralded as sterile, however, advances in culture-independent, next generation sequencing technology have revealed that this site harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most common non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy and diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to characterize genital tract lactobacilli to species-level taxonomy.MethodsSamples were interrogated for the presence of microbial DNA using two-step next generation sequencing technology to exploit the V5–V8 regions of the 16S rRNA gene and compared to standard speciation using qPCR.ResultsThe V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently encountered genital tract lactobacilli to allow accurate determination of relative abundance within the community, and speciation for several key community members without completing additional experimentation.ConclusionsNext-generation sequencing of clinical genital tract isolates is an effective method for high throughput identification to species-level of key Lactobacillus sp.IMPORTANCEHuman microbiome experiments, including the low biomass organs such as the upper genital tract, require the development of consensus protocols to ensure accurate comparison between such studies and our data forms an important foundation for future protocols.This paper provides evidence to support the selection of the V5-V8 regions of the 16S rRNA gene improved Lactobacillus speciation using next generation sequencing technology. The choice of variable region for broad-range amplification in microbiome studies is important due to preferential primer binding associated with some genera based on nucleotide sequence patterns. By utilising the V5-V8 region, multiple species of Lactobacillus can be characterised with relative confidence.

scholarly journals The Impact of Next Generation Sequencing in Cancer Research

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2928
Author(s):  
Katia Nones ◽  
Ann-Marie Patch
Keyword(s):  
Cancer Research ◽  
Next Generation Sequencing ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Next Generation ◽  
Parallel Sequencing ◽  
Next Generation Sequencing Ngs ◽  
The Impact ◽  
Generation Sequencing ◽  
Technical Revolution

Next generation sequencing (NGS) describes the technical revolution that enabled massively parallel sequencing of fragmented nucleic acids, thus making possible our current genomic understanding of cancers [...]

scholarly journals Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions

2010 ◽  
Vol 38 (22) ◽  
pp. e200-e200 ◽  
Author(s):  
Marcus J. Claesson ◽  
Qiong Wang ◽  
Orla O'Sullivan ◽  
Rachel Greene-Diniz ◽  
James R. Cole ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Microbiota Composition ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Generation Sequencing

scholarly journals Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

2017 ◽  
Vol 114 (18) ◽  
pp. 4733-4738 ◽  
Author(s):  
Austin K. Mattox ◽  
Yuxuan Wang ◽  
Simeon Springer ◽  
Joshua D. Cohen ◽  
Srinivasan Yegnasubramanian ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Clinical Samples ◽  
Low Frequencies ◽  
Parallel Sequencing ◽  
Molecular Barcoding ◽  
Bisulfite Treatment ◽  
Rare Mutations ◽  
Template Dna ◽  
Generation Sequencing

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

scholarly journals Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroaki Nakanishi ◽  
Katsumi Yoneyama ◽  
Masaaki Hara ◽  
Aya Takada ◽  
Kazuyuki Saito
Keyword(s):  
Animal Species ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
12S Rrna ◽  
Sequencing Analysis ◽  
Cytochrome Oxidase Subunit ◽  
Parallel Sequencing ◽  
Mitochondrial 12S Rrna ◽  
Crude Drugs ◽  
Mixed Crude

AbstractWe developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

scholarly journals Mixed culture metagenomics of the microbes making sour beer

2021 ◽  
Author(s):  
Renan Eugenio Araujo Piraine ◽  
Fabio Pereira Leivas Leite ◽  
Matthew L. Bochman
Keyword(s):  
Genetic Material ◽  
Rrna Gene ◽  
Internal Transcribed Spacer Region ◽  
Microbiome Analysis ◽  
Fungal Identification ◽  
Sample Composition ◽  
Fungal Dna ◽  
Mixed Microbial Cultures ◽  
The 16S Rrna Gene ◽  
Generation Sequencing

Mixed microbial cultures create sour beers, but many brewers do not know which microbes comprise their cultures. The objective of this work was to use deep sequencing to identify mi-croorganisms in sour beers brewed by spontaneous and non-spontaneous methods. Twenty samples were received from brewers, which were processed for microbiome analysis by next generation sequencing. For bacteria, primers were used to amplify the V3-V4 region of the 16S rRNA gene; fungal DNA detection was performed using primers to amplify the entire internal transcribed spacer region. The sequencing results were then used for taxonomy assignment, sample composition, and diversity analyses, as well as nucleotide BLAST searching. We identi-fied 60 genera and 140 species of bacteria, of which the most prevalent were Lactobacillus acetotol-erans, Pediococcus damnosus, and Ralstonia picketti/mannitolilytica. In fungal identification, 19 genera and 26 species were found, among which the most common yeasts were Brettanomyces bruxellen-sis and Saccharomyces cerevisiae. In some cases, genetic material from more than 60 microorgan-isms was found in a single sample. In conclusion, we were able to determine the microbiomes of various mixed cultures used to produce beer, providing useful information to better understand the sour beer fermentation process and brewing techniques.

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