Influence of sequencing depth on bacterial classification and abundance in bacterial communities

The effects of a subacute ruminal acidosis (SARA) challenge on the composition of epimural and mucosa-associated bacterial communities throughout the digestive tract were determined in eight non-lactating Holstein cows. Treatments included feeding a control diet containing 19.6% dry matter (DM) starch and a SARA-challenge diet containing 33.3% DM starch for two days after a 4-day grain step-up. Subsequently, epithelial samples from the rumen and mucosa samples from the duodenum, proximal, middle and distal jejunum, ileum, cecum and colon were collected. Extracted DNA from these samples were analyzed using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. Distinct clustering patterns for each diet existed for all sites. The SARA challenge decreased microbial diversity at all sites, with the exception of the middle jejunum. The SARA challenge also affected the relative abundances of several major phyla and genera at all sites but the magnitude of these effects differed among sites. In the rumen and colon, the largest effects were an increase in the relative abundance of Firmicutes and a reduction of Bacteroidetes. In the small intestine, the largest effect was an increase in the relative abundance of Actinobacteria. The grain-based SARA challenge conducted in this study did not only affect the composition and cause dysbiosis of epimural microbiota in the rumen, it also affected the mucosa-associated microbiota in the intestines. To assess the extent of this dysbiosis, its effects on the functionality of these microbiota must be determined in future.

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New Insights on the Zeugodacus cucurbitae (Coquillett) Bacteriome

Microorganisms ◽

10.3390/microorganisms9030659 ◽

2021 ◽

Vol 9 (3) ◽

pp. 659

Author(s):

Elias Asimakis ◽

Panagiota Stathopoulou ◽

Apostolis Sapounas ◽

Kanjana Khaeso ◽

Costas Batargias ◽

...

Keyword(s):

Relative Abundance ◽

Bacterial Communities ◽

High Throughput Sequencing ◽

Mutual Exclusion ◽

Bacterial Composition ◽

Operational Taxonomic Units ◽

Host Diet ◽

Dominant Bacteria ◽

High Degree

Various factors, including the insect host, diet, and surrounding ecosystem can shape the structure of the bacterial communities of insects. We have employed next generation, high-throughput sequencing of the 16S rRNA to characterize the bacteriome of wild Zeugodacus (Bactrocera) cucurbitae (Coquillett) flies from three regions of Bangladesh. The tested populations developed distinct bacterial communities with differences in bacterial composition, suggesting that geography has an impact on the fly bacteriome. The dominant bacteria belonged to the families Enterobacteriaceae, Dysgomonadaceae and Orbaceae, with the genera Dysgonomonas, Orbus and Citrobacter showing the highest relative abundance across populations. Network analysis indicated variable interactions between operational taxonomic units (OTUs), with cases of mutual exclusion and copresence. Certain bacterial genera with high relative abundance were also characterized by a high degree of interactions. Interestingly, genera with a low relative abundance like Shimwellia, Gilliamella, and Chishuiella were among those that showed abundant interactions, suggesting that they are also important components of the bacterial community. Such knowledge could help us identify ideal wild populations for domestication in the context of the sterile insect technique or similar biotechnological methods. Further characterization of this bacterial diversity with transcriptomic and metabolic approaches, could also reveal their specific role in Z. cucurbitae physiology.

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Optimized IGH Chain Sequencing for Detection of Very Low Frequency B Cell Malignancies from RNA or DNA Input

Blood ◽

10.1182/blood-2019-130525 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5777-5777

Author(s):

Timothy Looney ◽

Michelle Toro ◽

Geoffrey Lowman ◽

Jayde Chang ◽

Loni Pickle ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Lymphocytic Leukemia ◽

Sequencing Depth ◽

Sequencing Analysis ◽

Rarefaction Analysis ◽

Total Rna ◽

Fisher Scientific

Background High throughput sequencing (HTS) of rearranged TCRB and IGH chains has been demonstrated as a means to detect malignant T and B cells at a frequency as low as 10E-6. However, onerous input requirements (typically 20-30ug gDNA input over multiple library preparations) have impeded widespread adoption of 10E-6 as a threshold for minimal residual disease (MRD) translational research studies. Here we demonstrate an optimized highly multiplex PCR approach for amplifying IGH chains from fresh or FFPE-preserved RNA or DNA input material. Coupled with automated clonotyping and clonal lineage detection, we demonstrate detection of malignant B cell clones at a frequency of 10E-6 from a single library preparation. Methods Rearranged IGH chains were amplified using multiplex framework 3 and joining gene primers targeting all human IGH variable and joining gene alleles in the IMGT database (Oncomine IGH-SR assay). Libraries were generated from 25 or 100ng total RNA or 2ug gDNA derived from (1) peripheral blood leukocyte (PBL) or bone marrow (BM) spiked with Ramos B-cell cell line and (2) PBL or BM spiked with synthesized chronic lymphocytic leukemia (CLL) rearrangements from literature. Sequencing analysis was performed using the Gene Studio S5 and Ion Reporter to identify clonotypes, track clones across samples, and identify B cell clonal lineages. Clonal lineages were defined such that lineage members have a shared variable and joining gene identity, identical CDR3 lengths, and CDR3NT sequences within 85% similarity of other lineage members. Automated rarefaction analysis in Ion Reporter was used to determine optimal sequencing depth. Results Ramos and synthesized spike in controls were detected at a frequency of 10E-5 using 25ng of PBL total RNA and sequencing to 3M reads depth, and 10E-6 using 100ng input and 10M reads depth. gDNA-based libraries required 2ug and 3M reads depth to detect spike-in rearrangements at a frequency of 10E-5, while 10E-6 was achieved by combining the results from four 2ug gDNA libraries, each sequenced to 3M reads depth. Input and sequencing depth requirements were consistent across PBL or bone marrow derived libraries. Rarefaction analysis confirmed that the sequencing depth was appropriate for the targeted limit of detection. Conclusions These results demonstrate routine detection of B cell malignancy IGH chains at a frequency of 10E-6 using a limited amount of RNA or DNA material, comparing favorably to existing HTS-based approaches. We anticipate this approach to become a routine component of rare clone tracking applications including those involving B-ALL and CLL, particularly where sample material is limited or a low limit of detection is of paramount importance. Disclosures Looney: Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.

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A checklist of gilled mushrooms (Basidiomycota: Agaricomycetes) with diversity analysis in Hollongapar Gibbon Wildlife Sanctuary, Assam, India

Journal of Threatened Taxa ◽

10.11609/jott.1770.7.15.8272-8287 ◽

2015 ◽

Vol 7 (15) ◽

pp. 8272 ◽

Cited By ~ 1

Author(s):

Girish Gogoi ◽

Vipin Parkash

Keyword(s):

Species Richness ◽

Diversity Index ◽

Abundant Species ◽

Shannon Diversity Index ◽

Diversity Analysis ◽

Wildlife Sanctuary ◽

Shannon Diversity ◽

Number Of Species ◽

Simpson Diversity Index ◽

Richness Index

Hollongapar Gibbon Wildlife Sanctuary is comprised of five distinct compartments. A total of 138 species of gilled mushrooms belonging to 48 genera, 23 families, five orders of the class Agaricomycetes, division Basidiomycota, have been collected and analyzed. The order Agaricales was was found with the highest number of species (113), followed by Russulales (14), Polyporales (5), Cantharellales (4) and Boletales (2). The species Coprinellus disseminatus and Megacollybia rodmani have shown the highest (8.26) and the lowest density (0.05), respectively. A total of 24 species, e.g., Termitomyces albuminosus, Marasmius curreyi, Marasmiellus candidus, Leucocoprinus medioflavus, Mycena leaiana, Hygrocybe miniata, Collybia chrysoropha, Gymnopus confluens were common with frequency percentage of 11.9, whereas Megacollybia rodmani with less frequency percentage (2.4) was found only in few quadrates of the sanctuary. The highly abundant species were Termitomyces medius (91.7) and Coprinellus disseminatus (86.8), and less abundant species were Psilocybe wayanadensis (1.0) and Lepiota sp. (1.0) in the study site. The order of the species richness index (R) compartment wise was 2>3>4>5>1. Both the Shannon diversity index and Simpson diversity index of agarics was maximum (1.88, 0.98) in compartment 2, whereas minimum (1.72, 0.95) in compartment 1 and 5, respectively. Moreover, the compartment 2 was found very much similar with compartment 3 and very less similar with compartment 1.<div> </div>

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Ecological networks reveal contrasting patterns of bacterial and fungal communities in glacier-fed streams in Central Asia

10.7287/peerj.preprints.27887 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ze Ren ◽

Hongkai Gao

Keyword(s):

Bacterial Community ◽

Microbial Communities ◽

Central Asia ◽

Fungal Community ◽

Bacterial Communities ◽

Beta Diversity ◽

Hydrological Modeling ◽

Alpha Diversity ◽

Environmental Variation ◽

Fungal Communities

Bacterial and fungal communities in biofilms are important components in driving biogeochemical processes in stream ecosystems. Previous studies have well documented the patterns of bacterial alpha diversity in stream biofilms in glacier-fed streams, where, however, beta diversity of the microbial communities has received much less attention especially considering both bacterial and fungal communities. A focus on beta diversity can provide insights into the mechanisms driving community changes associated to large environmental fluctuations and disturbances, such as in glacier-fed streams. Moreover, modularity of co-occurrence networks can reveal more ecological and evolutionary properties of microbial communities beyond taxonomic groups. Here, integrating beta diversity and co-occurrence approach, we explored the network topology and modularity of the bacterial and fungal communities with consideration of environmental variation in glacier-fed streams in Central Asia. Combining results from hydrological modeling and normalized difference of vegetation index, this study highlighted that hydrological variables and vegetation status are major variables determining the environmental heterogeneity of glacier-fed streams. Bacterial communities formed a more complex and connected network, while the fungal communities formed a more clustered network. Moreover, the strong interrelations among the taxonomic dissimilarities of bacterial community and modules suggest they had common processes in driving diversity and taxonomic compositions across the heterogeneous environment. In contrast, fungal community and modules generally showed distinct driving processes to each other. Moreover, bacterial and fungal communities also had different driving processes. Furthermore, the variation of bacterial community and modules were strongly correlated with hydrological properties and vegetation status but not with nutrients, while fungal community and modules (except one module) were not associated with environmental variation. Our results suggest that bacterial and fungal communities had distinct mechanisms in structuring microbial networks, and environmental variation had strong influences on bacterial communities but not on fungal communities. The fungal communities have unique assembly mechanisms and physiological properties which might lead to their insensitive responses to environmental variations compared to bacterial communities. Overall, beyond alpha diversity in previous studies, these results add our knowledge that bacterial and fungal communities have contrasting assembly mechanisms and respond differently to environmental variation in glacier-fed streams.

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Ecological networks reveal contrasting patterns of bacterial and fungal communities in glacier-fed streams in Central Asia

10.7287/peerj.preprints.27887v1 ◽

2019 ◽

Author(s):

Ze Ren ◽

Hongkai Gao

Keyword(s):

Bacterial Community ◽

Microbial Communities ◽

Central Asia ◽

Fungal Community ◽

Bacterial Communities ◽

Beta Diversity ◽

Hydrological Modeling ◽

Alpha Diversity ◽

Environmental Variation ◽

Fungal Communities

Bacterial and fungal communities in biofilms are important components in driving biogeochemical processes in stream ecosystems. Previous studies have well documented the patterns of bacterial alpha diversity in stream biofilms in glacier-fed streams, where, however, beta diversity of the microbial communities has received much less attention especially considering both bacterial and fungal communities. A focus on beta diversity can provide insights into the mechanisms driving community changes associated to large environmental fluctuations and disturbances, such as in glacier-fed streams. Moreover, modularity of co-occurrence networks can reveal more ecological and evolutionary properties of microbial communities beyond taxonomic groups. Here, integrating beta diversity and co-occurrence approach, we explored the network topology and modularity of the bacterial and fungal communities with consideration of environmental variation in glacier-fed streams in Central Asia. Combining results from hydrological modeling and normalized difference of vegetation index, this study highlighted that hydrological variables and vegetation status are major variables determining the environmental heterogeneity of glacier-fed streams. Bacterial communities formed a more complex and connected network, while the fungal communities formed a more clustered network. Moreover, the strong interrelations among the taxonomic dissimilarities of bacterial community and modules suggest they had common processes in driving diversity and taxonomic compositions across the heterogeneous environment. In contrast, fungal community and modules generally showed distinct driving processes to each other. Moreover, bacterial and fungal communities also had different driving processes. Furthermore, the variation of bacterial community and modules were strongly correlated with hydrological properties and vegetation status but not with nutrients, while fungal community and modules (except one module) were not associated with environmental variation. Our results suggest that bacterial and fungal communities had distinct mechanisms in structuring microbial networks, and environmental variation had strong influences on bacterial communities but not on fungal communities. The fungal communities have unique assembly mechanisms and physiological properties which might lead to their insensitive responses to environmental variations compared to bacterial communities. Overall, beyond alpha diversity in previous studies, these results add our knowledge that bacterial and fungal communities have contrasting assembly mechanisms and respond differently to environmental variation in glacier-fed streams.

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IMMEDIATE AND LONG-TERM EFFECTS OF INSECTICIDE APPLICATION ON PARASITOIDS IN JACK PINE STANDS IN QUEBEC

The Canadian Entomologist ◽

10.4039/ent104263-2 ◽

1972 ◽

Vol 104 (2) ◽

pp. 263-270 ◽

Cited By ~ 3

Author(s):

Peter W. Price

Keyword(s):

Relative Abundance ◽

Jack Pine ◽

Ground Vegetation ◽

Long Term Effects ◽

Aerial Application ◽

Insecticide Application ◽

Number Of Species ◽

Adult Parasitoid ◽

Pine Stands

AbstractParasitoid populations were sampled before, and for 4 years following, an aerial application of the insecticide phosphamidon to control a sawfly outbreak. Adult parasitoid mortality was high because of spraying, but a reservoir of parasitoids in host cocoons remained to repopulate the treated areas. In moister sites the number of species decreased and their relative abundance changed, but moderate numbers of parasitoids remained 4 years after spraying. In a dry site with little ground vegetation, none of the species present before spraying remained by the fourth year.

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O-143 Characterization of vaginal and endometrial microbiome in patients with chronic endometritis (CE)

Human Reproduction ◽

10.1093/humrep/deab127.011 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

P LOZANO ◽

A Bernabeu ◽

B Lledó ◽

R Morales ◽

F I Aranda ◽

...

Keyword(s):

16S Rrna ◽

Embryo Transfer ◽

Relative Abundance ◽

Beta Diversity ◽

Alpha Diversity ◽

Shannon Index ◽

Small Sample ◽

Diagnostic Methods ◽

Vaginal Microbiome ◽

Chronic Endometritis

Abstract Study question Could vaginal and endometrial microbiome by sequencing 16S rRNA be comparable to classic diagnostic methods or immunohistochemistry CD138 for diagnosis of chronic endometritis? Summary answer A characteristic endometrial and vaginal microbiome is present in patients with chronic endometritis. An abnormal vaginal microbiome correlates with the presence of chronic endometritis. What is known already Chronic endometritis is a disease characterized by persistent inflammation of the endometrial lining. Currently, histopathological evaluation by immunohistochemistry CD138 marker is most common diagnostic method for CE. Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of pathogenic microorganisms associated with CE. The main bacteria at vaginal and endometrial level belong to genus Lactobacillus, producers of lactic acid that allows maintaining acidic pH of vagina and acts as barrier against pathogens. Investigations on the effect of an abnormal endometrial and vaginal microbiome could improve assisted reproductive technologies. Study design, size, duration This is a observational pilot study (60 patients and 120 samples). The study population consists of patients attending to our fertility clinic for frozen euploid embryo transfer (FET) from May 2017 to May 2019. Preimplantation Genetic Testing of aneuploidy (PGT-A) was performed at blastocyst stage using Veriseq (Illumina). The inclusion criteria to be meet by patients were: age between 18 and 50 years, own or donated oocytes and use of ICSI. Participants/materials, setting, methods Cohort study with sixty patients undergoing assisted reproductive treatment (TRA) with their own or donated gametes and PGT-A Vaginal and endometrial samples were taken in the cycle prior to embryo transfer. The vaginal and endometrial microbiome was analyzed by mass sequencing of the V3V4 region of 16S rRNA. Bioinformatics analysis was performed using QIIME2 and MicrobiomeAnalyst packages. Alpha, beta diversity and taxonomic characterization were compared with positive and negative CD138 groups for chronic endometritis (CE). Main results and the role of chance Different bacterial communities were detected when vaginal and endometrial samples were analyzed in patients with and without endometritis diagnosed with CD138 immunohistochemistry. In patients with endometritis, a higher alpha diversity index tendency was found in vaginal samples (p = 0.15 for the Shannon index) and significant differences in endometrial samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between the groups established as per the diagnosis of endometritis. Vaginal and endometrial samples from women with endometritis showed a microbiome pattern not dominated by Lactobacillus spp. Relative abundance analysis identified the genera Ralstonia and Gardnerella in endometrial sample, and the genera Streptoccoccus and Ureaplasma in vaginal sample of patients diagnosed with CD138 for endometritis. Comparing endometrial and vaginal samples CD138 positive diagnosed for endometritis, alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Relative abundance identified the genera Lactobacillus (p = 3.76E-4), Ralstonia (p = 8.19E-4), Delftia (p = 0.004) and Anaerobacillus (p = 0.004) in these sample groups. Limitations, reasons for caution The main limitation of this study is the small sample size. Larger studies including a higher number of samples are needed to confirm the different microbiome pattern observed at the vaginal and endometrial levels in correlation with chronic endometritis. The microbiome pattern has not been analyzed after treatment of CE. Wider implications of the findings Our findings suggest the existence of a characteristic vaginal and endometrial microbiota in patients with chronic endometritis. Different genera and species were identified in patients with and without endometritis depending on whether the sample was endometrial or vaginal. An abnormal vaginal microbiome appears to be strongly correlated with chronic endometritis. Trial registration number Not Applicable

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Endophytic bacterial communities of oilseed rape associate with genotype-specific resistance against Verticillium longisporum

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz188 ◽

2019 ◽

Vol 96 (1) ◽

Cited By ~ 1

Author(s):

Stefanie P Glaeser ◽

Iulian Gabur ◽

Hossein Haghighi ◽

Jens-Ole Bartz ◽

Peter Kämpfer ◽

...

Keyword(s):

Oilseed Rape ◽

Relative Abundance ◽

Bacterial Communities ◽

Quantitative Resistance ◽

Bacterial Community Composition ◽

Phylogenetic Group ◽

Rrna Gene ◽

Host Genotype ◽

Brassica Napus L ◽

Verticillium Longisporum

ABSTRACT Associations of endophytic bacterial community composition of oilseed rape (Brassica napus L.) with quantitative resistance against the soil-borne fungal pathogen Verticillium longisporum was assessed by 16S rRNA gene amplicon sequencing in roots and hypocotyls of four plant lines with contrasting genetic composition in regard to quantitative resistance reactions. The plant compartment was found to be the dominating driving factor for the specificity of bacterial communities in healthy plants. Furthermore, V. longisporum infection triggered a stabilization of phylogenetic group abundance in replicated samples suggesting a host genotype-specific selection. Genotype-specific associations with bacterial phylogenetic group abundance were identified by comparison of plant genotype groups (resistant versus susceptible) and treatment groups (healthy versus V. longisporum-infected) allowing dissection into constitutive and induced directional association patterns. Relative abundance of Flavobacteria, Pseudomonas, Rhizobium and Cellvibrio was associated with resistance/susceptibility. Relative abundance of Flavobacteria and Cellvibrio was increased in resistant genotypes according to their known ecological functions. In contrast, a higher relative abundance of Pseudomonas and Rhizobium, which are known to harbor many species with antagonistic properties to fungal pathogens, was found to be associated with susceptibility, indicating that these groups do not play a major role in genetically controlled resistance of oilseed rape against V. longisporum.

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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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