scholarly journals Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

2021 ◽  
Vol 9 (6) ◽  
pp. 1251
Author(s):  
Isabel Abellan-Schneyder ◽  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Mareike Wenning ◽  
Klaus Neuhaus
Keyword(s):  
Gene Sequencing ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Milk Samples ◽  
Rrna Gene Sequencing

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcusuberis, Streptococcusagalactiae, Streptococcusdysgalactiae, Escherichiacoli and Staphylococcusaureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

scholarly journals Genotype characterisation of Giardia duodenalis isolates from domestic and farm animals by SSU-rRNA gene sequencing

2004 ◽  
Vol 122 (3) ◽  
pp. 193-199 ◽  
Author(s):  
Federica Berrilli ◽  
David Di Cave ◽  
Claudio De Liberato ◽  
Alessia Franco ◽  
Paola Scaramozzino ◽  
...  
Keyword(s):  
Giardia Duodenalis ◽  
Gene Sequencing ◽  
Farm Animals ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Rrna Gene Sequencing

scholarly journals Impact of Bead-Beating Intensity on the Genus- and Species-Level Characterization of the Gut Microbiome Using Amplicon and Complete 16S rRNA Gene Sequencing

2021 ◽  
Vol 11 ◽  
Author(s):  
Bo Zhang ◽  
Matthew Brock ◽  
Carlos Arana ◽  
Chaitanya Dende ◽  
Nicolai Stanislas van Oers ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Bead Beating ◽  
Rrna Gene Sequencing

Bead-beating within a DNA extraction protocol is critical for complete microbial cell lysis and accurate assessment of the abundance and composition of the microbiome. While the impact of bead-beating on the recovery of OTUs at the phylum and class level have been studied, its influence on species-level microbiome recovery is not clear. Recent advances in sequencing technology has allowed species-level resolution of the microbiome using full length 16S rRNA gene sequencing instead of smaller amplicons that only capture a few hypervariable regions of the gene. We sequenced the v3-v4 hypervariable region as well as the full length 16S rRNA gene in mouse and human stool samples and discovered major clusters of gut bacteria that exhibit different levels of sensitivity to bead-beating treatment. Full length 16S rRNA gene sequencing unraveled vast species diversity in the mouse and human gut microbiome and enabled characterization of several unclassified OTUs in amplicon data. Many species of major gut commensals such as Bacteroides, Lactobacillus, Blautia, Clostridium, Escherichia, Roseburia, Helicobacter, and Ruminococcus were identified. Interestingly, v3-v4 amplicon data classified about 50% of Ruminococcus reads as Ruminococcus gnavus species which showed maximum abundance in a 9 min beaten sample. However, the remaining 50% of reads could not be assigned to any species. Full length 16S rRNA gene sequencing data showed that the majority of the unclassified reads were Ruminococcus albus species which unlike R. gnavus showed maximum recovery in the unbeaten sample instead. Furthermore, we found that the Blautia hominis and Streptococcus parasanguinis species were differently sensitive to bead-beating treatment than the rest of the species in these genera. Thus, the present study demonstrates species level variations in sensitivity to bead-beating treatment that could only be resolved with full length 16S rRNA sequencing. This study identifies species of common gut commensals and potential pathogens that require minimum (0-1 min) or extensive (4-9 min) bead-beating for their maximal recovery.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

scholarly journals Prevalence and molecular characterization of Cryptosporidium spp. in Père David’s deer (Elaphurus davidianus) in Jiangsu, China

2020 ◽  
Vol 29 (2) ◽  
Author(s):  
Si-Yang Huang ◽  
Yi-Min Fan ◽  
Yi Yang ◽  
Yi-Jun Ren ◽  
Jing-Zhi Gong ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Small Subunit ◽  
Basic Knowledge ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Cryptosporidium Spp ◽  
Père David’S Deer ◽  
Rrna Gene Sequencing

Abstract Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David’s deer. In this study, 137 fecal samples from Père David’s deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David’s deer in this area.

scholarly journals Species level resolution of female bladder microbiota from marker gene surveys

2020 ◽  
Author(s):  
Carter Hoffman ◽  
Nazema Y Siddiqui ◽  
Ian Fields ◽  
W. Thomas Gregory ◽  
Holly Simon ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
Variable Region ◽  
16S Rrna Gene Sequencing ◽  
Species Level ◽  
Reference Database ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Level Identification

AbstractThe human bladder contains bacteria in the absence of infection. Interest in studying these bacteria and their association with bladder conditions is increasing, but the chosen experimental method can limit the resolution of the taxonomy that can be assigned to the bacteria found in the bladder. 16S rRNA gene sequencing is commonly used to identify bacteria, but is typically restricted to genus-level identification. Our primary aim was to determine if accurate species-level identification of bladder bacteria is possible using 16S rRNA gene sequencing. We evaluated the ability of different classification schemes, each consisting of combinations of a 16S rRNA gene variable region, a reference database, and a taxonomic classification algorithm to correctly classify bladder bacteria. We show that species-level identification is possible, and that the reference database chosen is the most important component, followed by the 16S variable region sequenced.ImportanceSpecies-level information may deepen our understanding of associations between bladder microbiota and bladder conditions, such as lower urinary tract symptoms and urinary tract infections. The capability to identify bacterial species depends on large databases of sequences, algorithms that leverage statistics and available computer hardware, and knowledge of bacterial genetics and classification. Taken together, this is a daunting body of knowledge to become familiar with before the simple question of bacterial identity can be answered. Our results show the choice of taxonomic database and variable region of the 16S rRNA gene sequence makes species level identification possible. We also show this improvement can be achieved through the more careful application of existing methods and use of existing resources.

The smallest known heliozoans are the Erebor lineage (nom. clad. n.) inside Microheliella maris (Eukaryota, Diaphoretickes), with the amendation of M. maris diagnosis and description of Berkeleyaesol magnus gen. nov., comb. nov. (Eukaryota, incertae sedis)

2021 ◽  
Vol 71 (4) ◽  
Author(s):  
Yegor Shishkin ◽  
Daria Drachko ◽  
Vasily V. Zlatogursky
Keyword(s):  
New Genus ◽  
Gene Sequencing ◽  
Sister Group ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Morphometric Data ◽  
Free Living ◽  
Incertae Sedis ◽  
Rrna Gene Sequencing ◽  
Cell Body Size

A new strain of planktonic heliozoans (ZI172) belonging to the genus Microheliella (the sister group of Cryptista in Diaphoretickes), closely related to the only one known strain of Microheliella maris (CCAP 1945/1), was studied with light microscopy and SSU rRNA gene sequencing. Morphometric data obtained from 127 cells and based on 254 measurements showed that this strain represents the smallest heliozoan (1.66–3.42 µm, av. 2.56 µm) in diameter known to date and one of the smallest free-living eukaryotes. We also did morphometry for strain CCAP 1945/1. Its cell body size is 3.20–6.47 µm (av. 4.15 µm; n=141; m=282). The secondary structures of hairpin 15 of the SSU rRNA molecules were reconstructed for ZI172 and CCAP 1945/1 and they were compared The possible biochemical explanation for the smaller size of the ZI172 strain, which is smaller than the CCAP 1945/1 strain, is discussed, including all published electron micrographs of CCAP 1945/1. The necessary taxonomic work is also carried out. The diagnosis of Microheliella maris is amended and the new infraspecific clade Erebor is described to include ZI172. The measurements and systematics of the enigmatic heliozoan ‘Raphidiophrys’ magna O’Donoghue 1922 (non 1921; the biggest known heliozoan) are also discussed and it is transferred to the new genus Berkeleyaesol.

scholarly journals ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Isabel Abellan-Schneyder ◽  
Andrea Janina Schusser ◽  
Klaus Neuhaus
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Limiting Factor ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Dna Amounts

Abstract Background One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols. Results Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts. Conclusion Our improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

scholarly journals Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yoshiyuki Matsuo ◽  
Shinnosuke Komiya ◽  
Yoshiaki Yasumizu ◽  
Yuki Yasuoka ◽  
Katsura Mizushima ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Read

Abstract Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

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