Serra da Estrela PDO Cheese Microbiome as Revealed by Next Generation Sequencing

Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes’ milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3−V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01−1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

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Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

BMC Veterinary Research ◽

10.1186/s12917-021-02969-9 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Elton J. R. Vasconcelos ◽

Chayan Roy ◽

Joseph A. Geiger ◽

Kristina M. Oney ◽

Melody Koo ◽

...

Keyword(s):

Veterinary Medicine ◽

Next Generation Sequencing ◽

Complete Analysis ◽

Molecular Diagnostic ◽

Rrna Gene ◽

Spotted Fever Group ◽

Next Generation ◽

Vector Borne ◽

16 S Rrna ◽

Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

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Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

Microorganisms ◽

10.3390/microorganisms9010021 ◽

2020 ◽

Vol 9 (1) ◽

pp. 21

Author(s):

Abdul Ghafar ◽

Anson V. Koehler ◽

Ross S. Hall ◽

Charles G. Gauci ◽

Robin B. Gasser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Water Buffalo ◽

Hypervariable Region ◽

Small Subunit ◽

Rrna Gene ◽

Next Generation ◽

Small Subunit Rrna ◽

Tropical Theileriosis ◽

Ecological Zones ◽

Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

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Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

Foot & Ankle Orthopaedics ◽

10.1177/2473011420s00027 ◽

2020 ◽

Vol 5 (4) ◽

pp. 2473011420S0002

Author(s):

Yoonjung Choi ◽

Irvin Oh

Keyword(s):

Next Generation Sequencing ◽

False Positive ◽

Bacterial Genome ◽

Rrna Gene ◽

Next Generation ◽

Antibiotic Resistant ◽

Streptococcus Species ◽

Resistant Genes ◽

Standard Culture ◽

Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

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Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009779 ◽

2021 ◽

Vol 15 (10) ◽

pp. e0009779

Author(s):

Fakhriddin Sarzhanov ◽

Funda Dogruman-Al ◽

Monica Santin ◽

Jenny G. Maloney ◽

Ayse Semra Gureser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gastrointestinal Symptoms ◽

Molecular Methods ◽

Clinical Samples ◽

Rrna Gene ◽

Next Generation ◽

Dientamoeba Fragilis ◽

Significant Difference ◽

Generation Sequencing ◽

Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

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Bacterial abundance and community composition in green, brown and red water from intensive catfish (Clarias sp.) culture ponds in Yogyakarta, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220910 ◽

2021 ◽

Vol 22 (9) ◽

Author(s):

Karunia Adetera Nungki Wijayanti ◽

Indah Istiqomah ◽

Murwantoko Murwantoko

Keyword(s):

Next Generation Sequencing ◽

Bacterial Community ◽

16S Rrna ◽

Community Composition ◽

Water Samples ◽

Bacterial Abundance ◽

Rrna Gene ◽

Next Generation ◽

Aquaculture System ◽

Generation Sequencing

Abstract. Wijayanti KAN, Istiqomah I, Murwantoko. 2021. Bacterial abundance and community composition in green, brown and red water from intensive Catfish (Clarias sp.) culture ponds in Yogyakarta, Indonesia. Biodiversitas 22: 3677-3684. Catfish (Clarias sp.) is an important aquaculture commodity in Indonesia and cultured in an intensive system. Microorganisms play an important role in maintaining water quality of aquaculture system. The objective of this study was to determine the bacterial abundance and community composition of green, brown and red water collected from intensive catfish culture ponds in Yogyakarta using next-generation sequencing method. The water samples were collected from intensive catfish culture ponds with different colors, namely green, brown and red ponds located in Yogyakarta. The DNA from water samples was extracted using DNA extraction kit and used as template for 16S rRNA amplification. The V3-V4 hypervariable regions of the 16S rRNA gene were amplified apply for next-generation sequencing technology. This study could explore effectively the bacterial community in water samples. The bacterial communities in this catfish culture water showed higher bacterial richness compared to the other aquaculture system. The diversity of the green, brown and red catfish culture water ponds was similar with the number OTUs of the green, brown and red water samples, which were 1269; 1387 and 1323 OTUs respectively. The 694 OTUs (34.42%) were common core microbiomes in all catfish culture ponds, the 212 OTUs (10.51%) are present on green and brown water ponds, the 182 OTUs (9.02%) were on green and red water ponds, and the 183 OTUs (9.07%) were present on green and brown water ponds. However, the composition of the bacterial community was different. The most dominant phylum in green and brown water ponds was Proteobacteria with relative abundance in green water and brown water 71.6% and 47.0% respectively, whereas, the most dominant phylum in red water was Firmicutes (29.5%). The dominance of Firmicutes phylum in red water ponds may be caused by application of probiotic bacteria, the high organic content, and low oxygen concentration.

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Alcov: Estimating Variant of Concern Abundance from SARS-CoV-2 Wastewater Sequencing Data

10.1101/2021.06.03.21258306 ◽

2021 ◽

Author(s):

Isaac Ellmen ◽

Michael D.J. Lynch ◽

Delaney Nash ◽

Jiujun Cheng ◽

Jozef I. Nissimov ◽

...

Keyword(s):

Next Generation Sequencing ◽

Clinical Data ◽

Relative Abundance ◽

Community Level ◽

Next Generation ◽

Sequencing Data ◽

Complete Genomes ◽

Relative Abundances ◽

Wastewater Samples ◽

Generation Sequencing

Detection of SARS-CoV-2 in wastewater is an important strategy for community level surveillance. Variants of concern (VOCs) can be detected in the wastewater samples using next generation sequencing, however it can be challenging to determine the relative abundance of different VOCs since the reads cannot be assembled into complete genomes. Here, we present Alcov (abundance learning of SARS-CoV-2 variants), a tool that uses mutation frequencies in SARS-CoV-2 sequencing data to predict the distribution of VOC lineages in the sample. We used Alcov to predict the distributions of lineages from three wastewater samples which agreed well with clinical data. By predicting not just which VOCs are present, but their relative abundances in the population, Alcov extracts a more complete snapshot of the variants which are circulating in a community.

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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

10.1101/630004 ◽

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.

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What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?

Pathogens ◽

10.3390/pathogens11010034 ◽

2021 ◽

Vol 11 (1) ◽

pp. 34

Author(s):

Paula Santibáñez ◽

Concepción García-García ◽

Aránzazu Portillo ◽

Sonia Santibáñez ◽

Lara García-Álvarez ◽

...

Keyword(s):

Next Generation Sequencing ◽

Infective Endocarditis ◽

16S Rrna ◽

16S Rrna Gene ◽

Heart Valve ◽

Rrna Gene ◽

Microbiological Diagnosis ◽

Next Generation ◽

Targeted Ngs ◽

Generation Sequencing

Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore the potential utility of metataxonomics for improving microbiological diagnosis of IE. Here, next-generation sequencing (NGS) of the V3–V4 region of the 16S rRNA gene was performed in 27 heart valve tissues (18 natives, 5 intravascular devices, and 4 prosthetics) from 27 patients diagnosed with IE (4 of them with negative blood cultures). Metataxonomics matched with conventional diagnostic techniques in 24/27 cases (88.9%). The same bacterial family was assigned to 24 cases; the same genus, to 23 cases; and the same species, to 13 cases. In 22 of them, the etiological agent was represented by percentages > 99% of the reads and in two cases, by ~70%. Staphylococcus aureus was detected in a previously microbiological undiagnosed patient. Thus, microbiological diagnosis with 16S rRNA gene targeted-NGS was possible in one more sample than using traditional techniques. The remaining two patients showed no coincidence between traditional and 16S rRNA gene-targeted NGS microbiological diagnoses. In addition, 16S rRNA gene-targeted NGS allowed us to suggest coinfections that were supported by clinical data in one patient, and minority records also verified mixed infections in three cases. In our series, metataxonomics was valid for the identification of the causative agents, although more studies are needed before implementation of 16S rRNA gene-targeted NGS for the diagnosis of IE.

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Genomic constitution of diploid species of the genus Avena L. with A-genome according to the data of the next-generation sequencing (NGS)

Проблемы ботаники Южной Сибири и Монголии ◽

10.14258/pbssm.2021066 ◽

2021 ◽

Vol 20 (1) ◽

pp. 333-335

Author(s):

N. N. Nosov ◽

A. A. Gnutikov ◽

I. G. Loskutov ◽

E. V. Blinova ◽

A. V. Rodionov

Keyword(s):

Next Generation Sequencing ◽

Diploid Species ◽

Rrna Gene ◽

Next Generation ◽

Illumina Platform ◽

5.8S Rrna Gene ◽

A Genome ◽

5.8S Rrna ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

For diploid (2x) species with the A-genome, as well as for hexaploid (6x) from the genus Avena, a locus-specific next-generation sequencing (NGS) of the sequence of the region of the internal transcribed spacer ITS1 and the beginning of the 5.8S rRNA gene was carried out on the Illumina platform. The high diversity and heterogeneity of the genomes of diploid species are shown. It was revealed that the genomes of modern diploid oat species are relatively far removed from the hexaploid species. It was found that A. canariensis occupies an isolated position among other diploid species, and also takes only an insignificant role in the formation of hexaploid genomes.

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