Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions

High throughput sequencing has emerged as one of the most important techniques for characterizing microbial dynamics and revealing bacteria and host interactions. However, data interpretation using this technique is mainly based on relative abundance and ignores total bacteria load. In certain cases, absolute abundance is more important than compositional relative data, and interpretation of microbiota data based solely on relative abundance can be misleading. The available approaches for absolute quantification are highly diverse and challenging, especially for quantification in differing biological situations, such as distinguishing between live and dead cells, quantification of specific taxa, enumeration of low biomass samples, large sample size feasibility, and the detection of various other cellular features. In this review, we first illustrate the importance of integrating absolute abundance into microbiome data interpretation. Second, we briefly discuss the most widely used cell-based and molecular-based bacterial load quantification methods, including fluorescence spectroscopy, flow cytometry, 16S qPCR, 16S qRT-PCR, ddPCR, and reference spike-in. Last, we present a specific decision-making scheme for absolute quantification methods based on different biological questions and some of the latest quantitative methods and procedure modifications.

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Assessing soil bacterial community and dynamics by integrated high-throughput absolute abundance quantification

PeerJ ◽

10.7717/peerj.4514 ◽

2018 ◽

Vol 6 ◽

pp. e4514 ◽

Cited By ~ 25

Author(s):

Jun Lou ◽

Li Yang ◽

Haizhen Wang ◽

Laosheng Wu ◽

Jianming Xu

Keyword(s):

Bacterial Community ◽

High Throughput ◽

Relative Abundance ◽

High Throughput Sequencing ◽

Laboratory Strain ◽

Soil Bacterial Community ◽

Absolute Abundance ◽

Internal Reference ◽

Biodegradation Study ◽

Soil Bacterial

Microbial ecological studies have been remarkably promoted by the high-throughput sequencing approach with explosive information of taxonomy and relative abundance. However, relative abundance does not reflect the quantity of the microbial community and the inter-sample differences among taxa. In this study, we refined and applied an integrated high-throughput absolute abundance quantification (iHAAQ) method to better characterize soil quantitative bacterial community through combining the relative abundance (by high-throughput sequencing) and total bacterial quantities (by quantitative PCR). The proposed iHAAQ method was validated by an internal reference strain EDL933 and a laboratory strain WG5. Application of the iHAAQ method to a soil phenanthrene biodegradation study showed that for some bacterial taxa, the changes of relative and absolute abundances were coincident, while for others the changes were opposite. With the addition of a microbial activity inhibitor (NaN3), the absolute abundances of soil bacterial taxa, including several dominant genera of Bacillus, Flavobacterium, and Paenibacillus, decreased significantly, but their relative abundances increased after 28 days of incubation. We conclude that the iHAAQ method can offer more comprehensive information to reflect the dynamics of soil bacterial community with both relative and absolute abundances than the relative abundance from high-throughput sequencing alone.

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Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Scientific Reports ◽

10.1038/s41598-021-83318-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatsuhiko Hoshino ◽

Ryohei Nakao ◽

Hideyuki Doi ◽

Toshifumi Minamoto

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Correlation Coefficients ◽

Environmental Dna ◽

Digital Pcr ◽

Absolute Quantification ◽

Taqman Probe ◽

Individual Species ◽

Natural Environments ◽

Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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Taxonomic and Functional Characteristics of the Gill and Gastrointestinal Microbiota and Its Correlation with Intestinal Metabolites in NEW GIFT Strain of Farmed Adult Nile Tilapia (Oreochromis niloticus)

Microorganisms ◽

10.3390/microorganisms9030617 ◽

2021 ◽

Vol 9 (3) ◽

pp. 617

Author(s):

Zhenbing Wu ◽

Qianqian Zhang ◽

Yaoyao Lin ◽

Jingwen Hao ◽

Shuyi Wang ◽

...

Keyword(s):

Relative Abundance ◽

Oreochromis Niloticus ◽

Nile Tilapia ◽

High Throughput Sequencing ◽

Gas Chromatography Mass Spectrometry ◽

Gastrointestinal Mucosa ◽

Gastrointestinal Microbiota ◽

Linear Discriminant ◽

Intestinal Microbes ◽

Nile Tilapia Oreochromis Niloticus

The gill and gastrointestinal tract are primary entry routes for pathogens. The symbiotic microbiota are essential to the health, nutrition and disease of fish. Though the intestinal microbiota of Nile tilapia (Oreochromis niloticus) has been extensively studied, information on the mucosa-associated microbiota of this species, especially the gill and gastrointestinal mucosa-associated microbiota, is lacking. This study aimed to characterize the gill and gastrointestinal mucosa- and digesta-associated microbiota, as well as the intestinal metabolite profiles in the New Genetically Improved Farmed Tilapia (NEW GIFT) strain of farmed adult Nile tilapia by high-throughput sequencing and gas chromatography/mass spectrometry metabolomics. The diversity, structure, composition, and predicted function of gastrointestinal microbiota were significantly different across gastrointestinal regions and sample types (Welch t-test; p < 0.05). By comparing the mucosa- and digesta-associated microbiota, linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed that Pelomonas, Ralstoniapickettii, Comamonadaceae, and Staphylococcus were significantly enriched in the mucosa-associated microbiota, whereas many bacterial taxa were significantly enriched in the digesta-associated microbiota, including Chitinophagaceae, Cetobacterium, CandidatusCompetibacter, Methyloparacoccus, and chloroplast (LDA score > 3.5). Furthermore, Undibacterium, Escherichia–Shigella, Paeniclostridium, and Cetobacterium were dominant in the intestinal contents and mucosae, whereas Sphingomonasaquatilis and Roseomonasgilardii were commonly found in the gill and stomach mucosae. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analysis revealed that the predictive function of digesta-associated microbiota significantly differed from that of mucosa-associated microbiota (R = 0.8152, p = 0.0001). In addition, our results showed a significant interdependence between specific intestinal microbes and metabolites. Notably, the relative abundance values of several potentially beneficial microbes, including Undibacterium, Crenothrix, and Cetobacterium, were positively correlated with most intestinal metabolites, whereas the relative abundance values of some potential opportunistic pathogens, including Acinetobacter, Mycobacterium, Escherichia–Shigella, Paeniclostridium, Aeromonas, and Clostridiumsensustricto 1, were negatively correlated with most intestinal metabolites. This study revealed the characteristics of gill and gastrointestinal mucosa-associated and digesta-associated microbiota of farmed Nile tilapia and identified a close correlation between intestinal microbes and metabolites. The results serve as a basis for the effective application of targeted probiotics or prebiotics in the diet to regulate the nutrition and health of farmed tilapia.

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Urbanization pressures alter tree rhizosphere microbiomes

Scientific Reports ◽

10.1038/s41598-021-88839-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Carl L. Rosier ◽

Shawn W. Polson ◽

Vincent D’Amico ◽

Jinjun Kan ◽

Tara L. E. Trammell

Keyword(s):

Organic Matter ◽

Relative Abundance ◽

Tree Species ◽

High Throughput Sequencing ◽

Regional Climate ◽

Keystone Species ◽

Fagus Grandifolia ◽

Yellow Poplar ◽

Individual Tree ◽

Structural Formation

AbstractThe soil microbial community (SMC) provides critical ecosystem services including organic matter decomposition, soil structural formation, and nutrient cycling. Studies suggest plants, specifically trees, act as soil keystone species controlling SMC structure via multiple mechanisms (e.g., litter chemistry, root exudates, and canopy alteration of precipitation). Tree influence on SMC is shaped by local/regional climate effects on forested environments and the connection of forests to surrounding landscapes (e.g., urbanization). Urban soils offer an ideal analog to assess the influence of environmental conditions versus plant species-specific controls on SMC. We used next generation high throughput sequencing to characterize the SMC of specific tree species (Fagus grandifolia [beech] vs Liriodendron tulipifera [yellow poplar]) across an urban–rural gradient. Results indicate SMC dissimilarity within rural forests suggests the SMC is unique to individual tree species. However, greater urbanization pressure increased SMC similarity between tree species. Relative abundance, species richness, and evenness suggest that increases in similarity within urban forests is not the result of biodiversity loss, but rather due to greater overlap of shared taxa. Evaluation of soil chemistry across the rural–urban gradient indicate pH, Ca+, and organic matter are largely responsible for driving relative abundance of specific SMC members.

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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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Seasonal Variation Characteristics of Bacteria and Fungi in PM2.5 in Typical Basin Cities of Xi’an and Linfen, China

Atmosphere ◽

10.3390/atmos12070809 ◽

2021 ◽

Vol 12 (7) ◽

pp. 809

Author(s):

Sen Wang ◽

Wanyu Liu ◽

Jun Li ◽

Haotian Sun ◽

Yali Qian ◽

...

Keyword(s):

Seasonal Variation ◽

Relative Abundance ◽

Pathogenic Bacteria ◽

High Throughput Sequencing ◽

Fine Particulate Matter ◽

Ecological Functions ◽

Factors Affecting ◽

Bacterial Phyla ◽

Variation Characteristics ◽

Bacteria And Fungi

Microorganisms existing in airborne fine particulate matter (PM2.5) have key implications in biogeochemical cycling and human health. In this study, PM2.5 samples, collected in the typical basin cities of Xi’an and Linfen, China, were analyzed through high-throughput sequencing to understand microbial seasonal variation characteristics and ecological functions. For bacteria, the highest richness and diversity were identified in autumn. The bacterial phyla were dominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Metabolism was the most abundant pathway, with the highest relative abundance found in autumn. Pathogenic bacteria (Pseudomonas, Acinetobacter, Serratia, and Delftia) were positively correlated with most disease-related pathways. Besides, C cycling dominated in spring and summer, while N cycling dominated in autumn and winter. The relative abundance of S cycling was highest during winter in Linfen. For fungi, the highest richness was found in summer. Basidiomycota and Ascomycota mainly constituted the fungal phyla. Moreover, temperature (T) and sulfur dioxide (SO2) in Xi’an, and T, SO2, and nitrogen dioxide (NO2) in Linfen were the key factors affecting microbial community structures, which were associated with different pollution characteristics in Xi’an and Linfen. Overall, these results provide an important reference for the research into airborne microbial seasonal variations, along with their ecological functions and health impacts.

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Dynamics of Diversity and Abundance of Sulfonamide Resistant Bacteria in a Silt Loam Soil Fertilized by Compost

Antibiotics ◽

10.3390/antibiotics10060699 ◽

2021 ◽

Vol 10 (6) ◽

pp. 699

Author(s):

Hui Han ◽

Mohan Bai ◽

Yanting Chen ◽

Yali Gong ◽

Ming Wu ◽

...

Keyword(s):

High Throughput ◽

Relative Abundance ◽

High Throughput Sequencing ◽

Resistant Bacteria ◽

Arable Soils ◽

Soil Bacterial Communities ◽

Sulfonamide Resistance ◽

Loam Soil ◽

Abundance And Diversity ◽

Sulfonamide Resistance Genes

Although composting is effective in deactivating antibiotic substances in manure, the influence of compost fertilization on the occurrence and dissemination of antibiotic resistance in arable soils remains to be controversial. Herein, the abundance and diversity of two sulfonamide resistance genes (sul1 and sul2) in soil fertilized by compost spiked with two concentrations of sulfadiazine (1 and 10 mg kg−1) were studied intensively by qPCR and high throughput sequencing based on a two-month microcosm experiment. The concentration of sulfadiazine decreased rapidly after spiking from 25% at Day 1 to less than 2.7% at Day 60. Relative abundance of both sul1 and sul2 were significantly higher in soil amended with compost than the non-amended control at Day 1 and slightly decreased with incubation time except for sul2 in the S10 treatment. Soil bacterial communities were transiently shifted by compost fertilization regardless of the presence of sulfadiazine. Relative abundance of genera in three hubs positively interlinked with sul1 and sul2 were significantly higher in compost treated soil than the control at Day 1, 7 and 21, but not at Day 60. High throughput sequencing analyses revealed that most detected (>67% in relative abundance) sul1 and sul2 genotypes sharing >99% similarity with those found in gammaproteobacterial pathogens frequently were commonly present in compost and soil. These results indicated that compost fertilization might increase the abundance rather than diversity of sulfadiazine-resistant populations in soil, which may be facilitated by the presence of sulfadiazine.

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Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

Applied and Environmental Microbiology ◽

10.1128/aem.00560-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4757-4766 ◽

Cited By ~ 55

Author(s):

Caterina R. Giner ◽

Irene Forn ◽

Sarah Romac ◽

Ramiro Logares ◽

Colomban de Vargas ◽

...

Keyword(s):

Microbial Diversity ◽

High Throughput ◽

Relative Abundance ◽

Ribosomal Dna ◽

18S Rdna ◽

High Throughput Sequencing ◽

454 Sequencing ◽

Epifluorescence Microscopy ◽

Environmental Sequencing

ABSTRACTHigh-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescencein situhybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances.IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescencein situhybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.

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Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

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Abundance of Boeckella gracilis (Daday, 1902) (Copepoda, Calanoida) in seasonal coastal pools in the Araucania region (38°S, Chile)

Crustaceana ◽

10.1163/15685403-00003814 ◽

2018 ◽

Vol 91 (10) ◽

pp. 1211-1217

Author(s):

Patricio De los Ríos

Keyword(s):

Correlation Analysis ◽

Environmental Stress ◽

Relative Abundance ◽

Calanoid Copepod ◽

Inverse Correlation ◽

Inland Waters ◽

Absolute Abundance ◽

Significant Inverse Correlation

Abstract The presence of the calanoid copepod Boeckella gracilis (Daday, 1902) in Chilean seasonal pools has been only poorly studied as yet. The aim of the present study thus is to investigate the role of conductivity and temperature on the relative and absolute abundance of B. gracilis in seasonal coastal pools in the Araucania region (38°S, Chile). The results of correlation analysis revealed the presence of a significant inverse correlation between conductivity and relative abundance, whereas no significant correlations were found between conductivity and absolute abundance, between temperature and absolute abundance, and between temperature and relative abundance. These results agree partially with similar observations for mountain pools in the same region, but they would not agree with observations for calanoids of saline and subsaline inland waters in the northern and southern extremes of Chile. Considering this scenario, the species would show different populational responses to environmental stress in different situations, which phenomenon deserves to be studied more extensively and in more detail.

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