Academic literature on the topic '16S amplicon analysis'

Tropical reefs are highly diverse ecosystems, and reliable biomonitoring, through diet metabarcoding, is needed to understand present and future trophic relationships in this changing habitat. Several studies have assessed the reliability and effectiveness of single molecular markers; however, a cross-marker validation has rarely been performed. This study identified crucial properties for 12S rDNA, 16S rDNA and COI metabarcoding in tropical-reef piscivores (Plectropomus spp.). In addition, three new versatile primer sets for 16S were designed in silico for metabarcoding of reef fish. Results showed that COI was overall better at recovering true diversity because of a well-supported database. Second, optimal 16S amplicon sizes ranged between 160 and 440 base pairs for full diversity recovery, with increased species detection for the 270-base pairs region. Finally, blocking of predator-specific COI sequences was not equally effective in all host species, potentially introducing bias when diet compositions are directly compared. In conclusion, this novel study showed that marker success for prey identification is highly dependent on the reference database, taxonomic scope, DNA quality, amplicon length and sequencing platform. Results suggest that COI, complemented with 16S, yields the best outcome for diet metabarcoding in reef piscivores. Findings in this paper are relevant to other piscivores and other metabarcoding applications.

The taxonomic composition of microbial communities can be assessed using universal marker amplicon sequencing. The most common taxonomic markers are the 16S rDNA for bacterial communities and the internal transcribed spacer (ITS) region for fungal communities, but various other markers are used for barcoding eukaryotes. A crucial step in the bioinformatic analysis of amplicon sequences is the identification of representative sequences. This can be achieved using a clustering approach or by denoising raw sequencing reads. DADA2 is a widely adopted algorithm, released as an R library, that denoises marker-specific amplicons from next-generation sequencing and produces a set of representative sequences referred to as ‘Amplicon Sequence Variants’ (ASV). Here, we present Dadaist2, a modular pipeline, providing a complete suite for the analysis that ranges from raw sequencing reads to the statistics of numerical ecology. Dadaist2 implements a new approach that is specifically optimised for amplicons with variable lengths, such as the fungal ITS. The pipeline focuses on streamlining the data flow from the command line to R, with multiple options for statistical analysis and plotting, both interactive and automatic.

Background The full-length 16S rRNA sequencing can better improve the taxonomic and phylogenetic resolution compared to the partial 16S rRNA gene sequencing. The 16S-FAS-NGS (16S rRNA full-length amplicon sequencing based on a next-generation sequencing platform) technology can generate high-quality, full-length 16S rRNA gene sequences using short-read sequencers, together with assembly procedures. However there is a lack of a data analysis suite that can help process and analyze the synthetic long read data. Results Herein, we developed software named 16S-FASAS (16S full-length amplicon sequencing data analysis software) for 16S-FAS-NGS data analysis, which provided high-fidelity species-level microbiome data. 16S-FASAS consists of data quality control, de novo assembly, annotation, and visualization modules. We verified the performance of 16S-FASAS on both mock and fecal samples. In mock communities, we proved that taxonomy assignment by MegaBLAST had fewer misclassifications and tended to find more low abundance species than the USEARCH-UNOISE3-based classifier, resulting in species-level classification of 85.71% (6/7), 85.71% (6/7), 72.72% (8/11), and 70% (7/10) of the target bacteria. When applied to fecal samples, we found that the 16S-FAS-NGS datasets generated contigs grouped into 60 and 56 species, from which 71.62% (43/60) and 76.79% (43/56) were shared with the Pacbio datasets. Conclusions 16S-FASAS is a valuable tool that helps researchers process and interpret the results of full-length 16S rRNA gene sequencing. Depending on the full-length amplicon sequencing technology, the 16S-FASAS pipeline enables a more accurate report on the bacterial complexity of microbiome samples. 16S-FASAS is freely available for use at https://github.com/capitalbio-bioinfo/FASAS.

A discrepancy between cytology and bacterial culture methods is sometimes observed in canine otitis externa. The objective of this study was to compare results from cytology, bacterial culture and 16S amplicon profiling. Twenty samples from 16 dogs with chronic suppurative otitis externa were collected. A direct cytological evaluation was carried out during the consultations. Aerobic bacterial culture and susceptibility were performed by an external laboratory used in routine practice. For 16S amplicon profiling, DNA was extracted and the hypervariable segment V1–V3 of the 16S rDNA was amplified and then sequenced with a MiSeq Illumina sequence carried out by the Mothur software using the SILVA database. A good correlation between cytology and bacterial culture was observed in 60% of the samples. Some bacterial species revealed by bacterial culture were present with low relative abundance (<10%) in 16S amplicon profiling. Some bacterial species revealed by the 16S amplicon profiling analysis were not identified with culture; most of the time, the offending species was a Corynebacterium. To conclude, a careful interpretation of the results of bacterial culture should be made and always be in agreement with the cytology. The 16S amplicon profiling method appears to be a more sensitive method for detecting strains present in suppurative otitis but does not provide information on bacterial susceptibility.

With the increase in the number of households raising dogs and the reports of human-to-dog transmission of oral bacteria, concerns about dogs’ oral health and the need for oral hygiene management are increasing. In this study, the owners’ perceptions about their dogs’ oral health and the frequency of oral hygiene were determined along with the analysis of dog dental plaque bacteria through metagenomic amplicon sequencing so as to support the need for oral hygiene management for dogs. Although the perception of 63.2% of the owners about their dogs’ oral health was consistent with the veterinarian’s diagnosis, the owners’ oral hygiene practices regarding their dogs were very poor. The calculi index (CI) and gingiva index (GI) were lower in dogs who had their teeth brushed more than once a week (57.89%) than in dogs brushed less than once a month (42.10%); however, the difference was nonsignificant (CI: p = 0.479, GI: p = 0.840). Genomic DNA was extracted from dental plaque bacteria removed during dog teeth scaling, and metagenomic amplicons were sequenced. The 16S amplicons of 73 species were identified from among the plaque bacteria of the dogs. These amplicons were of oral disease-causing bacteria in humans and dogs. The 16S amplicon of Streptococcus mutans matched that of the human S. mutans, with type c identified as the main serotype. This result suggests that human oral bacteria can be transmitted to dogs. Therefore, considering the high frequency of contact between dogs and humans because of communal living and the current poor oral health of dogs, owners must improve the oral hygiene management of their dogs.

Bioinformatic tools for marker gene sequencing data analysis are continuously and rapidly evolving, thus integrating most recent techniques and tools is challenging. We present an R package for data analysis of 16S and ITS amplicons based sequencing. This workflow is based on several R functions and performs automatic treatments from fastq sequence files to diversity and differential analysis with statistical validation. The main purpose of this package is to automate bioinformatic analysis, ensure reproducibility between projects, and to be flexible enough to quickly integrate new bioinformatic tools or statistical methods. rANOMALY is an easy to install and customizable R package, that uses amplicon sequence variants (ASV) level for microbial community characterization. It integrates all assets of the latest bioinformatics methods, such as better sequence tracking, decontamination from control samples, use of multiple reference databases for taxonomic annotation, all main ecological analysis for which we propose advanced statistical tests, and a cross-validated differential analysis by four different methods. Our package produces ready to publish figures, and all of its outputs are made to be integrated in Rmarkdown code to produce automated reports.

ABSTRACTIn the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

GITDs are among the most common causes of death in adult and young horses in the United States (US). Previous studies have indicated a connection between GITDs and the equine gut microbiome. However, the low taxonomic resolution of the current microbiome sequencing methods has hampered the identification of specific bacterial changes associated with GITDs in horses. Here, we have compared TEHC, a new approach for 16S rRNA gene selection and sequencing, with conventional 16S rRNA gene amplicon sequencing for the characterization of the equine fecal microbiome. Both sequencing approaches were used to determine the fecal microbiome of four adult horses and one commercial mock microbiome. Our results show that TEHC yielded significantly more operational taxonomic units (OTUs) than conventional 16S amplicon sequencing when the same number of reads were used in the analysis. This translated into a deeper and more accurate characterization of the fecal microbiome when the samples were sequenced with TEHC according to the relative abundance analysis. Alpha and beta diversity metrics corroborated these findings and demonstrated that the microbiome of the fecal samples was significantly richer when sequenced with TEHC compared to 16S amplicon sequencing. Altogether, our study suggests that the TEHC strategy provides a more extensive characterization of the fecal microbiome of horses than the current alternative based on the PCR amplification of a portion of the 16S rRNA gene.

Resolution of the genetic heterogeneity of closely related insect species depends on the selection of reliable genetic markers derived from representative specimens. We report the results of a survey of genetic variability in nine species of black flies in the subgenusPsilopelmia Enderlein. Three regions of the mitochondrial genome and an amplicon including the internal transcribed spacer 1 of the nuclear ribosomal RNA gene cluster (ITS1) were amplified using the polymerase chain reaction (PCR), and the amplicons were examined for intraspecific and interspecific polymorphisms. Six of the seven Psilopelmia species that yielded PCR products in the ITS1 PCR reaction were found to generate products that were indistinguishable on the basis of size. Similarly, little interspecific variation was noted in the 16S rRNA amplicon among nine species of Psilopelmia assayed by heteroduplex analysis. In contrast, the remaining regions of the mitochondrial genome exhibited both intra- and inter-specific variation when analyzed by heteroduplex analysis. Information collected from the five amplicons could be employed to develop a classification scheme capable of distinguishing the nine species of Psilopelmia examined.

Nucleic acid sequencing can provide a detailed overview of microbial communities in comparison with standard plate-culture methods. Expansion of high-throughput sequencing (HTS) technologies and reduction in analysis costs has allowed for detailed exploration of various habitats with use of amplicon, metagenomics, and metatranscriptomics approaches. However, due to a capital cost of HTS platforms and requirements for batch analysis, genomics-based studies are still not being used as a standard method for the comprehensive examination of environmental or clinical samples for microbial characterization. This research project investigated the potential of a novel nanopore-based sequencing platform from Oxford Nanopore Technologies (ONT) for rapid and accurate analysis of various environmentally complex samples. ONT is an emerging company that developed the first-ever portable nanopore-based sequencing platform called MinIONTM. Portability and miniaturised size of the device gives an immense opportunity for de-centralised, in-field, and real-time analysis of environmental and clinical samples. Nonetheless, benchmarking of this new technology against the current gold-standard platform (i.e., Illumina sequencers) is necessary to evaluate nanopore data and understand its benefits and limitations. The focus of this study is on the evaluation of nanopore sequencing data: read quality, sequencing errors, alignment quality but also bacterial community structure. For this reason, mock bacterial community samples were generated, sequenced and analysed with use of multiple bioinformatics approaches. Furthermore, this study developed sophisticated library preparation and data analyses methods to enable high-accuracy analysis of amplicon libraries from complex microbial communities for sequencing on the nanopore platform. Besides, the best performing library preparation and data analyses methods were used for analysis of environmental samples and compared to high-quality Illumina metagenomics data. This work opens a new possibility for accurate, in-field amplicon analysis of complex samples with the use of MinIONTM and for the development of autonomous biosensing technology for culture-free detection of pathogenic and non-pathogenic microorganisms in water, soil, food, drinks or blood.

Les oligoéléments tels que le fer, le manganèse, le nickel et le cuivre jouent un rôle essentiel dans la croissance et le métabolisme des microorganismes procaryotes et eucaryotes phototrophes et non phototrophes dans l'océan, et influencent ainsi le cycle biogéochimique des principaux éléments. Alors que l'impact de la limitation en Fe sur les microorganismes phototrophes (phytoplancton) a été largement étudié, les liens entre les micronutriments et les procaryotes hétérotrophes restent jusqu'à présent peu étudiés. Cette thèse étudie le potentiel métabolique des microbes marins taxonomiquement diversifiés pour acquérir des métaux traces en utilisant une approche métagénomique et donne ainsi un aperçu de la contribution des taxons procaryotes au cycle des éléments mineurs et majeurs à des échelles temporelles et spatiales dans l'Océan Austral.L'objectif du chapitre 1 est d'étudier la stratégie des procaryotes pour acquérir différentes formes chimiques de Fe au cours de la saison. Sur la base d'échantillons obtenus par un échantillonneur autonome à distance, des observations métagénomiques saisonnières à haute résolution dans la région naturellement fertilisée en Fe au large des 'îles Kerguelen sont présentées. La dynamique saisonnière suggère un découplage temporel des besoins des procaryotes en Fe et en carbone organique pendant l'efflorescence printanière du phytoplancton et un accès concerté à ces ressources en fin d'été. Les stratégies écologiques donnent un aperçu de la manière dont le Fe pourrait façonner la composition de la communauté microbienne, avec des implications potentielles sur la transformation de la matière organique dans l'océan Austral.La question de savoir comment la disponibilité des métaux traces sélectionne les taxons procaryotes et les rétroactions potentielles des processus microbiens sur la distribution des métaux traces dans l'océan est abordée dans le chapitre 2. Les liens réciproques potentiels entre divers taxons procaryotes et Fe, Mn, Cu, Ni ainsi que l'utilisation apparente de l'oxygène (AOU) dans 12 masses d'eau bien définies du sud de l'océan Indien (SWINGS - South West Indian Ocean GEOTRACES GS02 Section cruise) ont été étudiés. L'analyse ‘Partial Least Square Regression' (PLSR) révèle que les masses d'eau sont associées à des vecteurs latents particuliers qui sont une combinaison de la distribution spatiale des taxons procaryotes, des éléments traces et de l'AOU. Cette approche fournit de nouvelles informations sur les interactions potentielles entre les taxons procaryotes et les métaux traces en relation avec la reminéralisation de la matière organique dans des masses d'eau distinctes de l'océan.Dans le chapitre 3, les gènes liés au transport des métaux-traces par les communautés procaryotes habitant les différentes masses d'eau échantillonnées au cours de la campagne SWINGS ont été étudiés. Ces résultats couvrent 42 échantillons prélevés de la surface jusqu'à la profondeur à 13 stations dans la zone subtropicale, subantarctique, frontale polaire et antarctique. L'abondance des gènes associés au transport du Fe, du Mn, du Ni et du Cu dans les fractions libres (<0,8 µm) et attachées aux particules (>0,8 µm) a été déterminée à la fois au niveau de la communauté et dans les ‘metagenome assembled genomes' (MAG). Les résultats révèlent des différences dans l'abondance normalisée (GPM) en fonction de la localisation géographique dans les eaux de surface et entre les masses d'eau en profondeur. Ces dernières étaient particulièrement prononcées pour les transporteurs de sidérophores, Mn, Ni et Cu. Chaque masse d'eau contenait un ensemble distinct de MAG abondants et ceux-ci se sont révélés différents dans leurs répertoires de transporteurs de métaux traces. Ces observations suggèrent que les métaux traces étudiés ici pourraient jouer des rôles distincts dans le métabolisme microbien dans différentes masses d'eau, avec des rétroactions potentielles sur leur cycle dans l'océan
Trace metals such as iron (Fe), manganese (Mn), nickel (Ni), and copper (Cu) play critical roles for the growth and metabolism of phototrophic and non-phototrophic prokaryotic and eukaryotic microorganisms in the ocean, and thereby influence the biogeochemical cycling of major elements. While the impact of Fe limitation on phototrophic microorganisms (phytoplankton) has been extensively investigated, the links between micronutrients and heterotrophic prokaryotes remains thus far poorly studied. This thesis investigates the metabolic potential of taxonomically diverse marine microbes to acquire trace metals using a metagenomics approach and thereby provides insights into the contribution of prokaryotic taxa to the cycling of minor and major elements on temporal and spatial scales in the Southern Ocean.The objective of Chapter 1 is to study the strategy of microbes to acquire different chemical forms of Fe under seasonally changing organic carbon requirements. Based on samples obtained by a remote autonomous sampler, high-resolution seasonal metagenomics observations from the naturally Fe-fertilized region off Kerguelen Island are presented. The results show pronounced, but distinct seasonal patterns in the abundance of genes implicated in the transport of different forms of Fe and organic substrates, of siderophore biosynthesis and carbohydrate active enzymes. The seasonal dynamics suggest a temporal decoupling in the prokaryotic requirements of Fe and organic carbon during the spring phytoplankton bloom and a concerted access to these resources after the summer bloom. Taxonomic assignments revealed differences in the prokaryotic groups harboring genes of a given Fe-related category and pronounced seasonal successions were observed. The ecological strategies provide insights on how Fe could shape microbial community composition with potential implications on organic matter transformations in the Southern Ocean.The question of how the availability of trace metals selects for prokaryotic taxa and the potential feedbacks of microbial processes on the trace metal distribution in the ocean is addressed in Chapter 2. The potential reciprocal links between diverse prokaryotic taxa and Fe, Mn, Cu, Ni as well as apparent oxygen utilization (AOU) across 12 well-defined water masses in the Southern Indian Ocean (SWINGS- South West Indian Ocean GEOTRACES GS02 Section cruise) was investigated. Partial Least Square Regression (PLSR) analysis reveals that the water masses are associated with particular latent vectors that are a combination of the spatial distribution of prokaryotic taxa, trace elements and AOU. This approach provides novel insights on the potential interactions between prokaryotic taxa and trace metals in relation to organic matter remineralization in distinct water masses of the ocean.In Chapter 3, genes related to trace-metal transport by the prokaryotic communities inhabiting the distinct water masses sampled during the SWINGS cruise were investigated. These results cover 42 samples collected from surface to depth at 13 stations in the Subtropical, Subantarctic, Polar Frontal and Antarctic zones. The abundance of genes associated with the transport of Fe, Mn, Ni and Cu in the free-living (<0.8 µm) and particle-attached fractions (>0.8 µm) were determined both at the community level as well as in metagenome assembled genomes (MAGs). The results reveal differences in the normalized abundance of these genes (GPM) depending on geographic location in surface waters, and among water masses at depth. These latter were particularly pronounced for transporters of siderophores, Mn, Ni and Cu. Each water mass contained a distinct set of abundant MAGs and these were found to differ in their repertoires of trace metal transporters. These observations suggest that the trace metals considered here could play distinct roles in microbial metabolism in different water masses, with potential feedbacks on their cycling in the ocean

This thesis describes the difference between the baseline composition of Diamondback moth (Plutella xylostella L.) insect colonies' gut microbiomes collected from field- and laboratory-reared populations. Results indicates that variation of gut bacterial community composition between individual insect influences baseline microbiome composition estimates at the population scale. The gut bacteria diversity and functional composition change in response to external challenges, and those changes reflect on insect development. This explains the need to discover the specific symbiotic gut bacteria that respond to environmental variables - such findings will be essential for controlling insect hosts, and this knowledge can be applied to develop insect pest management and control.

Mitochondrial DNA is an important tool for human identification and is used to differentiate between human and animal blood at the crime scene, because in extreme conditions nuclear DNA is severely destroyed while Mitochondrial DNA contains multiple copies (200–2000) per cell and resists harsh and more stable conditions. Seventy-two blood samples were collected from humans (Homo sapiens), sheep (Ovis aries), goats (Capra hircus), and cows (Bos taurus) (18 blood samples for each). All blood samples were withdrawn by a technician and 5 ml were aspirated using an aseptic technique and transferred to EDTA-Na2 tubes. They were mixed well and stored in a refrigerator. The collection took 2 weeks (May 15, 2019–May 30, 2019). All samples were collected from Al-Diwanyia city. The results of PCR testing revealed that the primer pairs were specific and non-specific products did not appear for all samples. The amplification of Homo sapiens mitochondrial DNA with primer pairs of other (Ovis aries, Capra hircus, and Bos taurus) and amplification of each with primer pairs of another genus gave negative results, and this is primary evidence for primer pair specificity. The amplicon of 16S rRNA gene of Homo sapiens was 1200 bp, Ovis aries was 1060 bp, Capra hircus was 820 bp, and Bos taurus was 1300 bp. The sequencing revealed that no cross-reactivity of designed primer pairs and the PCR assay based on the designed primer pairs will be simple, fast, sensitive, specific, and cost-effective. There is sensitivity, specificity, and accuracy in the designed species-specific primer pairs and applicability of the designed primer pairs in forensics to investigate blood spots or evidence belonging for human, sheep, goat, and cow.

With the recent advances in high throughput next-generation sequencing technologies and bioinformatics approach, gut microbiome research, especially in livestock species, has expanded immensely, elucidating the greatest potential to investigate the unacknowledged understanding of rumen microbiota in host physiology at the molecular level. The association of a complex aggregated community of microbes to host metabolism is of great importance due to their crucial participation in metabolic, immunological, and physiological tasks. The knowledge of this sophisticated network of a symbiotic association of gut microbiota to host organisms may lead to novel insights for improving health, enhancing production, and reducing the risk of disease progression in livestock species necessary to meet the demands of the human race. The full picture of microorganisms present in a particular area can be achieved with the help of culture-independent omics-based approaches. The integration of metagenomics, metatranscriptomics, metaproteomics, and meta-metabolomics technologies with systems biology emphasizes the taxonomic composition, identification, functional characterization, gene abundance, metabolic profiling, and phylogenetic information of microbial population along with the underlying mechanism for pathological processes and their involvement as probiotic. The rumen secretions or partially digested feed particles, as well as fecal samples, are generally employed for gut microbiome investigation. The 16S rRNA gene sequencing amplicon-based technology is the most employed technique for microbiome profiling in livestock species to date. The use of software and biological databases in the field of gut microbiome research gives an accurate in-depth analysis of the microbial population greatly.

Research objectives : Identify genetic potential and community structure of soil and rhizosphere microbial community structure as affected by treated wastewater (TWW) irrigation. This objective was achieved through the examination soil and rhizosphere microbial communities of plants irrigated with fresh water (FW) and TWW. Genomic DNA extracted from soil and rhizosphere samples (Minz laboratory) was processed for DNA-based shotgun metagenome sequencing (Green laboratory). High-throughput bioinformatics was performed to compare both taxonomic and functional gene (and pathway) differences between sample types (treatment and location). Identify metabolic pathways induced or repressed by TWW irrigation. To accomplish this objective, shotgun metatranscriptome (RNA-based) sequencing was performed. Expressed genes and pathways were compared to identify significantly differentially expressed features between rhizosphere communities of plants irrigated with FW and TWW. Identify microbial gene functions and pathways affected by TWW irrigation*. To accomplish this objective, we will perform a metaproteome comparison between rhizosphere communities of plants irrigated with FW and TWW and selected soil microbial activities. Integration and evaluation of microbial community function in relation to its structure and genetic potential, and to infer the in situ physiology and function of microbial communities in soil and rhizospere under FW and TWW irrigation regimes. This objective is ongoing due to the need for extensive bioinformatics analysis. As a result of the capabilities of the new PI, we have also been characterizing the transcriptome of the plant roots as affected by the TWW irrigation and comparing the function of the plants to that of the microbiome. *This original objective was not achieved in the course of this study due to technical issues, especially the need to replace the American PIs during the project. However, the fact we were able to analyze more than one plant system as a result of the abilities of the new American PI strengthened the power of the conclusions derived from studies for the 1ˢᵗ and 2ⁿᵈ objectives. Background: As the world population grows, more urban waste is discharged to the environment, and fresh water sources are being polluted. Developing and industrial countries are increasing the use of wastewater and treated wastewater (TWW) for agriculture practice, thus turning the waste product into a valuable resource. Wastewater supplies a year- round reliable source of nutrient-rich water. Despite continuing enhancements in TWW quality, TWW irrigation can still result in unexplained and undesirable effects on crops. In part, these undesirable effects may be attributed to, among other factors, to the effects of TWW on the plant microbiome. Previous studies, including our own, have presented the TWW effect on soil microbial activity and community composition. To the best of our knowledge, however, no comprehensive study yet has been conducted on the microbial population associated BARD Report - Project 4662 Page 2 of 16 BARD Report - Project 4662 Page 3 of 16 with plant roots irrigated with TWW – a critical information gap. In this work, we characterize the effect of TWW irrigation on root-associated microbial community structure and function by using the most innovative tools available in analyzing bacterial community- a combination of microbial marker gene amplicon sequencing, microbial shotunmetagenomics (DNA-based total community and gene content characterization), microbial metatranscriptomics (RNA-based total community and gene content characterization), and plant host transcriptome response. At the core of this research, a mesocosm experiment was conducted to study and characterize the effect of TWW irrigation on tomato and lettuce plants. A focus of this study was on the plant roots, their associated microbial communities, and on the functional activities of plant root-associated microbial communities. We have found that TWW irrigation changes both the soil and root microbial community composition, and that the shift in the plant root microbiome associated with different irrigation was as significant as the changes caused by the plant host or soil type. The change in microbial community structure was accompanied by changes in the microbial community-wide functional potential (i.e., gene content of the entire microbial community, as determined through shotgun metagenome sequencing). The relative abundance of many genes was significantly different in TWW irrigated root microbiome relative to FW-irrigated root microbial communities. For example, the relative abundance of genes encoding for transporters increased in TWW-irrigated roots increased relative to FW-irrigated roots. Similarly, the relative abundance of genes linked to potassium efflux, respiratory systems and nitrogen metabolism were elevated in TWW irrigated roots when compared to FW-irrigated roots. The increased relative abundance of denitrifying genes in TWW systems relative FW systems, suggests that TWW-irrigated roots are more anaerobic compare to FW irrigated root. These gene functional data are consistent with geochemical measurements made from these systems. Specifically, the TWW irrigated soils had higher pH, total organic compound (TOC), sodium, potassium and electric conductivity values in comparison to FW soils. Thus, the root microbiome genetic functional potential can be correlated with pH, TOC and EC values and these factors must take part in the shaping the root microbiome. The expressed functions, as found by the metatranscriptome analysis, revealed many genes that increase in TWW-irrigated plant root microbial population relative to those in the FW-irrigated plants. The most substantial (and significant) were sodium-proton antiporters and Na(+)-translocatingNADH-quinoneoxidoreductase (NQR). The latter protein uses the cell respiratory machinery to harness redox force and convert the energy for efflux of sodium. As the roots and their microbiomes are exposed to the same environmental conditions, it was previously hypothesized that understanding the soil and rhizospheremicrobiome response will shed light on natural processes in these niches. This study demonstrate how newly available tools can better define complex processes and their downstream consequences, such as irrigation with water from different qualities, and to identify primary cues sensed by the plant host irrigated with TWW. From an agricultural perspective, many common practices are complicated processes with many ‘moving parts’, and are hard to characterize and predict. Multiple edaphic and microbial factors are involved, and these can react to many environmental cues. These complex systems are in turn affected by plant growth and exudation, and associated features such as irrigation, fertilization and use of pesticides. However, the combination of shotgun metagenomics, microbial shotgun metatranscriptomics, plant transcriptomics, and physical measurement of soil characteristics provides a mechanism for integrating data from highly complex agricultural systems to eventually provide for plant physiological response prediction and monitoring. BARD Report