PSXIV-15 Metagenomic sequencing of rumen microorganisms of cattle fed a corn stover-based diet

Abstract Worldwide, there is a need to discover new microorganisms that efficiently degrade lignocellulosic complexes that would help to improve the digestibility of low-quality agricultural byproducts. The aim of the study was to evaluate the effects of a corn stover-based diet (CSD) on rumen bacteria. Ruminal fluid of 6 Holstein cows (595 ± 96 kg) was collected during two periods. During first period, animals were consuming a diet based on corn silage and oat hay (DB), mineral premix and water ad libitum (50:50, DM). In second period, animals were provided a CSD (100% DM), mineral premix and water ad libitum for 45 days. Ruminal fluid was collected through esophageal tube, filtered and stored at -80°C until DNA extraction. Rumen microorganisms were identified by sequencing the 16SrRNA gene using the Illumina Miseq platform and primers for V3 and V4 regions. Data were analyzed by QIIME 1.9. Analysis of variance was performed for a completely randomized design using the MIXED procedure of SAS 9.1. The taxonomic affiliation showed that both populations were mainly composed of Firmicutes, Bacteroidetes and Proteobacteria. The most abundant bacteria species in both diets were Ruminococcus flavefaciens, Prevotella copri, Prevotella ruminicola, Fibrobacter succinogenes, Bacillus coagulans, Bacteroides uniformis and Selenomonas ruminantium. Feeding a CSD, increased the relative abundance of Prevotella ruminicola (from 6.1 to 20.9%, P < 0.01), Streptococcus luteciae (from 0.05 to 0.78%, P < 0.01), Clostridium aminophilum (0.45 to 3.1%, P < 0.01), Selenomonas ruminantium (5.2 to 21.8%, P < 0.02) and Pantoea agglomerans (0.7 to 3.9%, P < 0.01) and decreased Propionibacterium acnes (0.7 to 0.1%, P < 0.02) and Bacteroides ovatus (0.9 to 0.1%, P < 0.01). Feeding cattle with a diet with a more lignified forage like CSD led to the proliferation of bacteria such as Prevotella ruminicula, Streptococcus luteciae, Clostridium aminophilum, Selenomonas ruminantium and Pantoea agglomerans.

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PSIX-6 Gastrointestinal microbiome of calves fed solid diets containing different levels and sources of NDF

Journal of Animal Science ◽

10.1093/jas/skaa278.729 ◽

2020 ◽

Vol 98 (Supplement_4) ◽

pp. 418-419

Author(s):

Gercino F Virgínio Júnior ◽

Milaine Poczynek ◽

Ana Paula Silva ◽

Ariany Toledo ◽

Amanda Cezar ◽

...

Keyword(s):

Illumina Miseq ◽

Diversity Indices ◽

Dairy Calves ◽

Rrna Gene ◽

Gastrointestinal Microbiome ◽

Fecal Microbiome ◽

Miseq Platform ◽

Holstein Calves ◽

Ad Libitum ◽

Different Levels

Abstract Different levels and sources of NDF can modify the gastrointestinal microbiome. This study evaluated 18 Holstein calves housed in not-bedded suspended individual cages and fed one of three treatments: 22NDF - conventional starter containing 22% NDF (n = 7); 31NDF - starter with 31% NDF, replacing part of the corn by soybean hull (n = 6); and 22NDF+H - conventional starter with 22% NDF plus coast-cross hay ad libitum (n = 5). All animals received 4 L of milk replacer daily (24% CP; 18.5% fat; diluted to 12.5% solids), divided into two meals, being weaned at 8th week of age. After weaning, animals were housed in tropical shelters, fed with the respective solid diet and coast-cross hay ad libitum for all treatments. To evaluate the microbiome, ruminal fluid samples were collected using a modified Geishauser oral probe at weeks 2, 4, 6, 8 and 10, two hours after the morning feeding, and fecal samples were collected at birth (0) and at weeks 1, 2, 4, 8 and 10. The microbial community was determined by sequencing V3 and V4 region amplicons of the 16S rRNA gene that was amplified by PCR and sequenced by the Illumina MiSeq platform. Ruminal microbiome had no differences in diversity for the effects of weeks, treatments or interaction of both factors (Table 1). In feces, the diversity indices and evenness were higher for 22NDF+H when compared to 22NDF, with no difference for 31NDF. All indices were significantly affected by calves age. At birth, calves had the greatest diversity and richness. Week 1 and 2 had less evenness and diversity. Bacteroidota, Firmicutes_A and Firmicutes_C were the most abundant phylum in rumen and feces. The supply of hay was only effective in modifying the fecal microbiome of dairy calves, suggesting a resilience in the ruminal microbiome.

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DNA extraction from concrete v2

10.17504/protocols.io.b2i5qcg6 ◽

2021 ◽

Author(s):

Anders Kiledal ◽

Julia A Maresca

Keyword(s):

Dna Extraction ◽

Dental Plaque ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Metagenomic Sequencing ◽

Sample Analysis ◽

Larger Sample ◽

Laboratory Contamination ◽

Biomass Sample ◽

Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

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Assessment of metagenomic Nanopore and Illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples

Eurosurveillance ◽

10.2807/1560-7917.es.2018.23.50.1800228 ◽

2018 ◽

Vol 23 (50) ◽

Cited By ~ 40

Author(s):

Liana E. Kafetzopoulou ◽

Kyriakos Efthymiadis ◽

Kuiama Lewandowski ◽

Ant Crook ◽

Dan Carter ◽

...

Keyword(s):

Illumina Miseq ◽

Clinical Samples ◽

Whole Genome ◽

Metagenomic Sequencing ◽

Front Line ◽

Genome Sequences ◽

Dengue Viruses ◽

Virus Identification ◽

Reverse Transcription Pcr ◽

Oxford Nanopore

Background The recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. Aim To investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples. Methods We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study. Results Direct metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. Conclusions This work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.

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Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples

10.1101/355560 ◽

2018 ◽

Cited By ~ 2

Author(s):

Liana E. Kafetzopoulou ◽

Kyriakos Efthymiadis ◽

Kuiama Lewandowski ◽

Ant Crook ◽

Dan Carter ◽

...

Keyword(s):

Dengue Virus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

Clinical Samples ◽

Whole Genome ◽

Metagenomic Sequencing ◽

Front Line ◽

Genome Sequences ◽

Oxford Nanopore

AbstractThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION to study the feasibility of whole genome sequencing from clinical samples containing chikungunya or dengue virus, two of the most important arboviruses. Direct metagenomic sequencing of nucleic acid extracts from serum and plasma without viral enrichment allowed for virus and coinfection identification, subtype determination and in the majority of cases elucidated complete or near-complete genomes adequate for phylogenetic analysis. This work demonstrates that metagenomic whole genome sequencing is feasible for over 90% and 80% of chikungunya and dengue virus PCR-positive patient samples respectively. It confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health.

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Heterogeneity of Soil Bacterial and Bacteriophage Communities in Three Rice Agroecosystems and Potential Impacts On Nutrient Cycling

10.21203/rs.3.rs-1179635/v1 ◽

2022 ◽

Author(s):

Yajiao Wang ◽

Yu Liu ◽

Yuxing Wu ◽

Nan Wu ◽

Wenwen Liu ◽

...

Keyword(s):

Community Structure ◽

Nutrient Cycling ◽

Soil Bacteria ◽

Southern China ◽

Eastern China ◽

Soil Microbial Communities ◽

Illumina Miseq ◽

Metagenomic Sequencing ◽

Soil Bacterial Diversity ◽

Soil Bacterial

Abstract Background: As genetic entities infecting and replicating only in bacteria, bacteriophages can regulate the community structure and functions of their host bacteria, but they are often overlooked because of their relatively low abundance. The ecological roles of bacteriophages in aquatic and forest environments have been widely explored, but those in agroecosystems remains limited. Here, we used metagenomic sequencing to analyze the diversity and interactions of bacteriophages and their host bacteria in soils from three typical rice agroecosystems in China: double cropping in Guangzhou, southern China, rice–wheat rotation cropping in Nanjing, eastern China and early maturing single cropping in Jiamusi, northeastern China. Bacteriophages were isolated and their functions on soil nitrogen cycling and effect on soil bacterial community structure were verified in pot inoculation experiments and Illumina MiSeq sequencing.Results: Soil bacterial and viral diversity and functions varied among the three agroecosystems. Genes detected in communities from the three agroecosystems were associated with typical functions; soil bacteria in Jiamusi were significantly enriched in genes related to carbohydrate metabolism, in Nanjing with xenobiotics biodegradation and metabolism, and in Guangzhou with virulence factors and scarce in secondary metabolite biosynthesis, which might lead to a significant occurrence of rice bacterial diseases. In the three ecosystems, 368 species of virus were detected. Notably, over-represented auxiliary carbohydrate-active enzyme (CAZyme) genes were identified in the viruses, which might assist host bacteria in metabolizing carbon, and 67.43% of these genes were present in Jiamusi. In bacteriophage isolation and inoculation experiments, Enterobacter bacteriophage-NJ reduced the nitrogen fixation capacity of soil by lysing N-fixing host bacteria and changed the soil bacterial diversity and community structure.Conclusions: Our results showed that diversity and function of paddy soil bacteria and viruses varied in the three agroecosystems. Soil bacteriophages can affect nutrient cycling by expressing auxiliary metabolic genes (AMGs) and lysing the host bacteria that are involved in biogeochemical cycles. These findings form a basis for better understanding bacterial and bacteriophage diversity in different rice agroecosystems, laying a solid foundation for further studies of soil microbial communities that support ecofriendly production of healthy rice.

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Metagenome Techniques for Detection of Pathogens Causing Ocular Infection

Reports ◽

10.3390/reports4010006 ◽

2021 ◽

Vol 4 (1) ◽

pp. 6

Author(s):

Tatsuhiko Kobayashi ◽

Takashi Suzuki ◽

Yukinobu Okajima ◽

Kotaro Aoki ◽

Yoshikazu Ishii ◽

...

Keyword(s):

Illumina Miseq ◽

Metagenomic Analysis ◽

Ocular Infection ◽

Fungal Culture ◽

Metagenomic Sequencing ◽

Viral Pathogens ◽

Metagenome Analysis ◽

Ocular Infections ◽

Shotgun Metagenomic Sequencing ◽

Corneal Scraping

Metagenomic analysis is the comprehensive study of DNA using clinical specimens of organisms including bacteria, fungi, and viruses. In this study, we investigated the efficacy of metagenomic analysis for diagnosing ocular infections, including 11 keratitis cases, four iridocyclitis cases, and one endophthalmitis case. Corneal scraping, aqueous humor, and vitreous humor, were collected respectively. Ocular specimens were used for bacterial and fungal culture, and PCR for detecting viral DNA. Shotgun metagenomic sequencing for 150 bases of single end was performed by Illumina MiSeq® System. Sequence was retrieved from the database at NCBI using a MegaBLAST search. Since Propionibacterium spp. are commensal bacteria found at the ocular surface, they were excluded from analysis. Six cases (37.5%) were positive for culture or PCR. Metagenome techniques revealed that 9 cases (56.3%) included genomes of organisms that were considered pathogenic in specimens. Five cases (31.3%) possessed genomes of organisms like themselves that were detected by culture and PCR. Six cases (37.5%) were negative for culture, PCR, and metagenome analysis. Moreover, viral pathogens (HSV-1, 2 cases; and VZV, 1 case) were detected by only metagenome analysis. Metagenome analysis using an ocular sample can detect microbial genome comprehensively, and viral pathogens, which were not detected by conventional examination.

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Perilla frutescens Leaf Alters the Rumen Microbial Community of Lactating Dairy Cows

Microorganisms ◽

10.3390/microorganisms7110562 ◽

2019 ◽

Vol 7 (11) ◽

pp. 562 ◽

Cited By ~ 4

Author(s):

Zhiqiang Sun ◽

Zhu Yu ◽

Bing Wang

Keyword(s):

Ph Value ◽

Block Design ◽

Control Diet ◽

Antioxidant Properties ◽

Illumina Miseq ◽

Perilla Frutescens ◽

Herbaceous Plant ◽

Ruminal Fluid ◽

Ruminal Ph ◽

Microbial Dna

Perilla frutescens (L.) Britt., an annual herbaceous plant, has antibacterial, anti-inflammation, and antioxidant properties. To understand the effects of P. frutescens leaf on the ruminal microbial ecology of cattle, Illumina MiSeq 16S rRNA sequencing technology was used. Fourteen cows were used in a randomized complete block design trial. Two diets were fed to these cattle: a control diet (CON); and CON supplemented with 300 g/d P. frutescens leaf (PFL) per cow. Ruminal fluid was sampled at the end of the experiment for microbial DNA extraction. Overall, our findings revealed that supplementation with PFL could increase ruminal fluid pH value. The ruminal bacterial community of cattle was dominated by Bacteroidetes, Firmicutes, and Proteobacteria. The addition of PFL had a positive effect on Firmicutes, Actinobacteria, and Spirochaetes, but had no effect on Bacteroidetes and Proteobacteria compared with the CON. The supplementation with PFL significantly increased the abundance of Marvinbryantia, Acetitomaculum, Ruminococcus gauvreauii, Eubacterium coprostanoligenes, Selenomonas_1, Pseudoscardovia, norank_f__Muribaculaceae, and Sharpea, and decreased the abundance of Treponema_2 compared to CON. Eubacterium coprostanoligenes, and norank_f__Muribaculaceae were positively correlated with ruminal pH value. It was found that norank_f__Muribaculaceae and Acetitomaculum were positively correlated with milk yield, indicating that these different genera are PFL associated bacteria. This study suggests that PFL supplementation could increase the ruminal pH value and induce shifts in the ruminal bacterial composition.

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Potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis

Future Cardiology ◽

10.2217/fca-2018-0088 ◽

2019 ◽

Vol 15 (6) ◽

pp. 411-424

Author(s):

Wai-Sing Chan ◽

Chun-Hang Au ◽

Henry Chi-Ming Leung ◽

Dona N Ho ◽

Dinghua Li ◽

...

Keyword(s):

Infective Endocarditis ◽

Heart Valves ◽

Illumina Miseq ◽

Metagenomic Sequencing ◽

Potential Utility ◽

Bacterial Genomes ◽

Bartonella Quintana ◽

Replacement Surgery ◽

Valvular Replacement ◽

Etiologic Diagnosis

Aim: To explore potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis (IE) caused by fastidious bacteria. Materials & methods: Plasma and heart valves of two patients, who were diagnosed with IE caused by Bartonella quintana and Propionibacterium species, were sequenced by using Illumina MiSeq and Nanopore MinION. Results: For patient 1, B. quintana was detected in the plasma pool collected 4 days before valvular replacement surgery. For patient 2, Propionibacterium sp. oral taxon 193 was detected in the plasma sample collected on hospital day 1. Nearly complete bacterial genomes (>98%) were retrieved from resected heart valves of both patients, enabling detection of antibiotic resistance-associated features. Real-time sequencing of heart valves identified both pathogens within the first 16 min of sequencing runs. Conclusion: Metagenomic sequencing may be a helpful supplement to IE diagnostic workflow, especially when conventional tests fail to yield a diagnosis.

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