DNA/RNA extraction and qPCR protocol to assess bacterial abundance in the spongeHalichondria panicea v1

2021 ◽  
Author(s):  
Lara Schmittmann ◽  
Lucia Pita
Keyword(s):  
16S Rrna ◽  
Bacterial Abundance ◽  
Rna Extraction ◽  
Amplicon Sequencing ◽  
Halichondria Panicea ◽  
16S Rrna Amplicon Sequencing ◽  
Dna And Rna ◽  
Sponge Halichondria Panicea

This protocol summarizes experience and recommendations regarding DNA and RNA extractions from the sponge Halichondria panicea and qPCR to quantify bacterial abundance. We have used both bacterial and sponge DNA and RNA for subsequent qPCR. Further, bacterial 16S rRNA amplicon sequencing was performed on DNA and sponge transcriptomes were sequenced successfully from extracted RNA.

scholarly journals DNA and RNA-SIP reveal Nitrospira spp. as key drivers of nitrification in groundwater-fed biofilters

2019 ◽  
Author(s):  
Arda Gülay ◽  
Jane Fowler ◽  
Karolina Tatari ◽  
Bo Thamdrup ◽  
Hans-Jørgen Albrechtsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Ammonia Oxidation ◽  
Sequence Similarity ◽  
Sodium Chlorate ◽  
Amplicon Sequencing ◽  
Stable Isotope Probing ◽  
Ammonium Oxidation ◽  
16S Rrna Sequence ◽  
16S Rrna Amplicon Sequencing ◽  
Dna And Rna

AbstractNitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier metagenomics, amoA-amplicon, and amoA-qPCR based investigations of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira, and we hypothesized that these non-classical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subject to a continuous supply of NH4+ or NO2− in the presence of 13C-HCO3− (labelled) or 12C-HCO3− (unlabelled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonium oxidation in the biofilter community. A total of 78 (8 in RNA-SIP and 70 in DNA-SIP) and 96 (25 in RNA-SIP and 71 in DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3− uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobactera, and Acidovorax under conditions that favoured ammonium oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia oxidizing taxa was not detected in the SIP fractions. This study provides the first in-situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome.

scholarly journals Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2414
Author(s):  
Laura Sanjulián ◽  
Alexandre Lamas ◽  
Rocío Barreiro ◽  
Alberto Cepeda ◽  
Cristina A. Fente ◽  
...  
Keyword(s):  
Breast Milk ◽  
16S Rrna ◽  
Human Milk ◽  
Alpha Diversity ◽  
Amplicon Sequencing ◽  
Maternal Body Mass Index ◽  
16S Rrna Amplicon Sequencing ◽  
Spanish Women ◽  
Calcium Magnesium ◽  
Abundant Genus

The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

Advantage of 16S rRNA amplicon sequencing in Helicobacter pylori diagnosis

Helicobacter ◽  
2021 ◽  
Author(s):  
Boldbaatar Gantuya ◽  
Hashem B. El Serag ◽  
Batsaikhan Saruuljavkhlan ◽  
Dashdorj Azzaya ◽  
Takashi Matsumoto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing

scholarly journals Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing

2021 ◽  
Vol 9 (7) ◽  
pp. 1525
Author(s):  
Can Akpolat ◽  
Ana Beatriz Fernández ◽  
Pinar Caglayan ◽  
Baris Calli ◽  
Meral Birbir ◽  
...  
Keyword(s):  
16S Rrna ◽  
Salt Lake ◽  
Amplicon Sequencing ◽  
Deep Zone ◽  
Biotechnological Potential ◽  
16S Rrna Amplicon Sequencing ◽  
Physico Chemical ◽  
Potential Applications ◽  
A Minor ◽  
Tuz Lake

Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum, Haloarcula, Halorhabdus, Natronomonas, Halosimplex, Halomicrobium, Halorubrum, Halonotius, Halolamina, Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum, Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications.

16S rRNA Amplicon Sequencing for Metagenomics

2018 ◽  
pp. 135-161
Author(s):  
Henrik Christensen ◽  
Anna Jasmine Andersson ◽  
Steffen Lynge Jørgensen ◽  
Josef Korbinian Vogt
Keyword(s):  
16S Rrna ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing

scholarly journals Dramatic differences in gut bacterial densities help to explain the relationship between diet and habitat in rainforest ants

2017 ◽  
Author(s):  
Jon G Sanders ◽  
Piotr Lukasik ◽  
Megan E Frederickson ◽  
Jacob A Russell ◽  
Ryuichi Koga ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Diversity ◽  
Tropical Rainforest ◽  
Amplicon Sequencing ◽  
Sequencing Data ◽  
Lowland Tropical Forest ◽  
16S Rrna Amplicon Sequencing ◽  
Microbial Symbionts ◽  
Microbial Symbiosis ◽  
Diversity Profiles

AbstractAbundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative PCR as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results help to resolve a longstanding question in tropical rainforest ecology, and have broad implications for the interpretation of sequence-based surveys of microbial diversity.

scholarly journals Evaluation of the microbial community structure of potable water samples from occupied and unoccupied buildings using16S rRNA amplicon sequencing

2020 ◽  
Author(s):  
Kimothy L Smith ◽  
Howard A Shuman ◽  
Douglas Findeisen
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Water Samples ◽  
Ad Hoc ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
The Other ◽  
Water Usage ◽  
16S Rrna Amplicon Sequencing ◽  
Oxford Nanopore

AbstractWe conducted two studies of water samples from buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location with matched water sample types. One of the buildings had been continuously occupied, and the other substantially vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings. Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in Study 1 and 8.3% of samples in Study 2.SynopsisComparison of microbial community composition in the plumbing of occupied and unoccupied buildings during the COVID-19 pandemic shutdown.

scholarly journals Microbial insights from Antarctic and Mediterranean shallow-water bone-eating worms

Polar Biology ◽  
2020 ◽  
Vol 43 (10) ◽  
pp. 1605-1621 ◽  
Author(s):  
Olivia H. Hewitt ◽  
Cristina Díez-Vives ◽  
Sergi Taboada
Keyword(s):  
16S Rrna ◽  
Shallow Water ◽  
Host Species ◽  
Heterotrophic Bacteria ◽  
Amplicon Sequencing ◽  
Body Tissue ◽  
16S Rrna Amplicon Sequencing ◽  
Degrading Bacteria ◽  
Contrasting Habitats ◽  
Root Tissues

Abstract Bone-eating worms of the genus Osedax (Annelida, Siboglinidae) form unique holobionts (functional entity comprising host and associated microbiota), highly adapted to inhabit bone tissue of marine vertebrates. These gutless worms have developed nutritional symbioses housing intracellular, horizontally acquired, heterotrophic bacteria hypothesised to harness nutrients from organic compounds, sequestered within the bone. Despite previous efforts, critical mechanisms mediating activity and acquisition of diverse bacterial assemblages remain unclear. Using 16S rRNA amplicon sequencing, we performed detailed taxonomic and predicted functional analyses shedding light on the microbial communities of two shallow-water Osedax species (Osedax deceptionensis and Osedax ‘mediterranea’) from contrasting habitats (Antarctic and Mediterranean Sea), in two tissue types (roots and palps). Comparative assessments between host species revealed distinct microbial assemblages whilst, within host species and body tissue, relative symbiont frequencies retained high variability. We reported relatively high abundances of microbes previously classified as primary endosymbionts, Ribotype 1 (order Oceanospirillales), and diverse likely secondary epibionts warranting further exploration as recurrent Osedax associates. Surprisingly, O. ‘mediterranea’ exhibited relatively low abundance of Oceanospirillales, but increased abundance of other potentially hydrocarbon degrading bacteria from the family Alteromonadaceae. We hypothesise the presence of functionally similar, non-Oceanospirillales primary endosymbionts within O. ‘mediterranea’. Functional metagenomic profiling (using 16S rRNA sequences) predicted broad metabolic capabilities, encompassing relatively large abundances of genes associated with amino acid metabolism. Comparative analyses between host body tissue communities highlighted several genes potentially providing critical functions to the Osedax host or that confer adaptations for intracellular life, housed within bone embedded host root tissues.

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