Next-generation Amplicon Sequencing Research Articles

Characterization of the bacterial microbiomes of social amoebae and exploration of the roles of host and environment on microbiome composition.

As predators of bacteria, amoebae select for traits that allow bacteria to become symbionts by surviving phagocytosis and exploiting the eukaryotic intracellular environment. Soil-dwelling social amoebae can help us answer questions about the natural ecology of these amoeba-bacteria symbioses along the pathogen-mutualist spectrum. Our objective was to characterize the natural bacterial microbiome of phylogenetically and morphologically diverse social amoeba species using next-generation sequencing of 16S rRNA amplicons directly from amoeba fruiting bodies. We found six phyla of amoeba-associated bacteria: Proteobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, Firmicutes, and Acidobacteria. The most common associates of amoebae were classified to order Chlamydiales and genus Burkholderia-Caballeronia-Paraburkholderia. These bacteria were present in multiple amoeba species across multiple locations. While there was substantial intraspecific variation, there was some evidence for host specificity and differentially abundant taxa between different amoeba hosts. Amoebae microbiomes were distinct from the microbiomes of their soil habitat, and soil pH affected amoeba microbiome diversity. Alpha-diversity was unsurprisingly lower in amoebae samples compared with soil, but beta-diversity between amoebae samples was higher than between soil samples. Further exploration of social amoebae microbiomes may help us understand the roles of bacteria, host, and environment on symbiotic interactions and microbiome formation in basal eukaryotic organisms.

Microbiological investigations of two thermal baths in Budapest, Hungary. Report: effect of bathing and pool operation type on water quality.

In Hungary, which is famous for its thermal baths, according to the regulations, waters are investigated in hygienic aspects with standard cultivation methods. In the present study, two thermal baths were investigated (the well and three different pool waters in both) using cultivation methods, taxon-specific polymerase chain reactions (PCRs), multiplex PCRs and next-generation amplicon sequencing. Mainly members of the natural microbial community of the well waters and bacteria originating from the environment were detected but several opportunistic pathogenic taxa, e.g., Pseudomonas aeruginosa, P. stutzeri, Acinetobacter johnsoni, Acinetobacter baumanni, Moraxella osloensis, Microbacterium paraoxydans, Legionella spp., Stenotrophomonas maltophilia and Staphylococcus aureus were revealed by the applied methods. Pools with charging-unloading operation had higher microscopic cell counts, colony-forming unit (CFU) counts, number of cocci, P. aeruginosa and S. aureus compared to the recirculation systems. Bacteria originating from human sources (e.g., skin) were identified in the pool waters with less than 1% relative abundance, and their presence was sporadic in the pools. Comparing the microbiological quality of the pools based on the first sampling time and the following four months' period it was revealed that recirculation operation type has better water quality than the charging-unloading pool operation from a hygienic point of view.

An application of compositional data analysis to multiomic time-series data.

Compositional data analysis (CoDA) methods have increased in popularity as a new framework for analyzing next-generation sequencing (NGS) data. CoDA methods, such as the centered log-ratio (clr) transformation, adjust for the compositional nature of NGS counts, which is not addressed by traditional normalization methods. CoDA has only been sparsely applied to NGS data generated from microbial communities or to multiple ‘omics’ datasets. In this study, we applied CoDA methods to analyze NGS and untargeted metabolomic datasets obtained from bacterial and fungal communities. Specifically, we used clr transformation to reanalyze NGS amplicon and metabolomics data from a study investigating the effects of building material type, moisture and time on microbial and metabolomic diversity. Compared to analysis of untransformed data, analysis of clr-transformed data revealed novel relationships and stronger associations between sample conditions and microbial and metabolic community profiles.

Inoculation of Ensifer fredii strain LP2/20 immobilized in agar results in growth promotion and alteration of bacterial community structure of Chinese kale planted soil

In our former research, we succeeded in using agar, alginate, and perlite as immobilization materials to maintain long-term survival of the inoculant, Ensifer fredii LP2/20, in a controlled glasshouse. Therefore the information on the establishment and activity of the inoculant to promote plant growth under field conditions, the effects of the inoculant on the soil microbial communities and specific microbial taxa, and the association between the inoculant and soil elements merit further studies. Here, we found that agar was the most suitable material that supported the establishment of the inoculant under field conditions. RNA-based analysis showed that E. fredii LP2/20 immobilized in agar was still metabolically active at day 50 after being introduced into soil. Inoculation of E. fredii LP2/20 immobilized in agar conferred the highest plant dry weight (up to 89.94%) and all plant elements including total N (9.55%), P (17.94%), K (68.42%), Ca (39.77%), Mg (30.76%), Fe (29.85%), and Zn (22.44%). Inoculation of E. fredii LP2/20 immobilized in agar increased soil chemicals including soil organic matter (99.02%), total N (272.48%), P (31.75%), K (52.74%), Fe (51.06%), and Zn (63.10%). High-throughput next-generation sequencing of bacterial 16S rRNA amplicons showed that the Proteobacteria, Acidobacteria, Bacteroidetes, and Firmicutes were dominant phyla in Chinese kale field soil. Inoculation of E. fredii LP2/20 significantly affected the soil bacterial community structure by decreasing total bacterial richness and diversity. The numbers of alpha- and gamma-Proteobacteria were significantly increased while the number of delta-Proteobacteria was significantly decreased due to E. fredii LP2/20 establishment. Soil total P, K, and Ca and soil pH were the important factors that shaped the soil bacterial community composition.

Characterization of prevalence and genetic subtypes of Blastocystis sp. in wild and domestic Suidae of central Italy aided by amplicon NGS.

Blastocystis spp. is a common single-celled intestinal symbiont, comprising several genetic subtypes (ST) and transmissible by animal-to-animal, human-to-human, animal-to-human and, possibly, human-to-animal routes. This work was designed to explore the presence of Blastocystis in sympatric domestic and wild suids and their ability to carry zoonotic STs, in a condition of widespread opportunity to come in contact with the microorganism through their shared water and food resources, and other carriers. We sampled 42 and 37 stool samples from wild boars and domestic pigs, respectively. STs were first identified by PCR followed by Sanger sequencing. Sequences represented in double-band PCR products or in Sanger chromatograms displaying multiple peaks, were resolved by next generation sequencing (NGS). Twenty-six (61.9%) wild boar and 26 (70.2%) pig samples were PCR-positive, respectively. ST3, ST5 and ST15 were found in 3.8%, 38.4% and 80.8% of the positive wild boars and 11.5%, 88.5%, 11.5% of the positive pigs, respectively. ST1 was found only in pigs (3.8%). STs 5 and 15 were common in both groups of animals, but in reversed proportions, suggesting preferential colonization. We found significantly different ST distributions among wild boars and domestic pigs. This might indicate that lifestyle differences between the two populations influence their risk for contracting certain subtypes, or that ST5 and ST15 can colonize preferentially wild or domestic animals. Based on the STs described here, wild boars and domestic pigs can act as reservoirs with zoonotic potential. The ability of suids to carry zoonotic STs appears to be higher when using NGS than Sanger sequencing, and resolution of complex sequencing profiles is imperative before excluding the presence of STs of human concern.

Root microbiome changes with root branching order and root chemistry in peach rhizosphere soil

The plant root system influences plant growth and development due to its phenotypic, physiological, metabolomic, and microbiomic traits. Broadly speaking, it is characterized by primary (stem-attached large), secondary (primary-attached medium), and fine (secondary-attached hair-like) roots. The role of root branching order and categories (fine, medium, and large) in influencing microbial communities in the rhizosphere and root environments is not clear. We studied whether and how different root categories influence the composition of root and rhizosphere microbial communities in young peach trees. Using next-generation 16S rRNA amplicon sequencing (V3–V4 region), we profiled the microbial communities of roots and rhizosphere environments from phylum to species-level taxonomies. We demonstrated that different root categories showed a unique microbial community composition. Interestingly, fine or small roots recruited more diverse and species-rich microbial communities, very likely due to their better mineral contents (e.g., Mo, Fe, Mn, S, Zn, Cu, K, and P) and exposure with soil, though more research is needed to establish these relationships. Small roots and their rhizosphere environments showed a higher abundance of important bacterial taxa (e.g., Bradyrhizobium, Pseudomonas, Streptomyces, Burkholderia, Sphingomonas) that are previously known to play important roles in soil disease suppressiveness, plant growth, nutrient fixation, solubilization, and cycling. We suggest that linking microbial communities and their functions to root branching order and categories may enhance our understanding of rhizospheric interactions, soil disease suppressiveness, and their role in tree fruit performance.

Evaluation of molecular inversion probe versus TruSeq® custom methods for targeted next-generation sequencing.

Resolving the genetic architecture of painful neuropathy will lead to better disease management strategies. We aimed to develop a reliable method to re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. In this study, we compared sensitivity, specificity, targeting efficiency, performance and cost effectiveness of Molecular Inversion Probes-Next generation sequencing (MIPs-NGS) and TruSeq® Custom Amplicon-Next generation sequencing (TSCA-NGS). Capture probes were designed to target nine sodium channel genes (SCN3A, SCN8A-SCN11A, and SCN1B-SCN4B). One hundred sixty-six patients with diabetic and idiopathic neuropathy were tested by both methods, 70 patients were validated by Sanger sequencing. Sensitivity, specificity and performance of both techniques were comparable, and in agreement with Sanger sequencing. The average targeted regions coverage for MIPs-NGS was 97.3% versus 93.9% for TSCA-NGS. MIPs-NGS has a more versatile assay design and is more flexible than TSCA-NGS. The cost of MIPs-NGS is >5 times cheaper than TSCA-NGS when 500 or more samples are tested. In conclusion, MIPs-NGS is a reliable, flexible, and relatively inexpensive method to detect genetic variations in a large cohort of patients. In our centers, MIPs-NGS is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients.

The Milk Microbiota of the Spanish Churra Sheep Breed: New Insights into the Complexity of the Milk Microbiome of Dairy Species.

Simple SummaryIn the last decade, the number of studies focused on the study of the microbiota of different tissues, organs, or physiological fluids has considerably increased. The milk of dairy species is an important and continuous source of commensal, mutualistic and potentially probiotic bacteria. Second-generation sequencing technologies have been applied to characterise the milk microbiota of dairy cows, whereas the study of the sheep milk microbiota is scarce. In the present study, we aimed to explore the bacterial diversity and composition of milk samples from the Churra sheep breed, a rustic autochthonous breed from the region of Castilla y León (Spain). Moreover, this study tries to clarify the complex bacterial composition of sheep milk comparing the results presented here with previous research on the milk microbiota of the Assaf sheep breed. This assessment has shown that the milk microbiota of ewes from one flock of the Assaf breed is more diverse than the milk microbiota reported here for two different flocks of Churra sheep. The study also provides a step into a better understanding of the link between the bacterial milk composition in these two sheep breeds and somatic cell count, an indicator trait of subclinical mastitis resistance in dairy sheep.Milk from healthy animals has classically been considered a sterile fluid. With the development of massively parallel sequencing and its application to the study of the microbiome of different body fluids, milk microbiota has been documented in several animal species. In this study, the main objective of this work was to access bacterial profiles of healthy milk samples using the next-generation sequencing of amplicons from the 16S rRNA gene to characterise the milk microbiome of the Churra breed. A total of 212 samples were collected from two Churra dairy farms with a different management system. The core milk microbiota in Churra ewes includes lesser genera (only two taxa: Staphylococcus and Escherichia/Shigella) than studies reported in other dairy species or even in a previous study in Assaf sheep milk. We found that diversity values in the two flocks of Churra breed were lower than the diversity of the milk microbiota in Assaf. The non-metric multidimensional scaling (NMDS) ordination using Bray-Curtis distance separates samples based on their microbiota composition. The information reported here might be used to understand the complex issue of milk microbiota composition.

Impact of Temperature, Nutrients and Heavy Metals on Bacterial Diversity and Ecosystem Functioning Studied by Freshwater Microcosms and High-Throughput DNA Sequencing.

Microbial communities are fundamental components in freshwater, and community shifts in ecosystem structure are indicative of changing environmental conditions. This study aimed at investigating the influence of key environmental parameters on bacterial diversity and ecosystem functioning (i.e. organic matter breakdown) in laboratory freshwater microcosms. The effects of varying temperatures (5, 20 and 35°C), nutrients (representing low, medium and high urbanization) and heavy metals Copper (Cu) and Zinc (Zn) on bacterial diversity and organic matter (OM) breakdown were studied by using leaf bags and capsules filled with polycaprolactonediol-2000 (PCP-2000), respectively. The leaf-associated bacterial diversity was determined by next-generation sequencing of SSU rRNA gene amplicons. The results showed that bacterial diversity increased at high temperature (35°C) with more operational taxonomic units (OTUs) as compared to medium (20°C) or low (5°C) temperatures, whereas nutrient variation had fewer effects on the bacterial community structure. In contrast, the presence of heavy metals, especially high concentrations (100μM) of Cu, reduced the number of OTUs in the leaf-associated bacterial community. The higher temperatures and nutrient levels accelerated PCP-2000 breakdown rate, but this was impeded by a high concentration (100μM) of Cu in the short term, though no effect of Zn on breakdown rate was observed. The overall results indicate that temperature and variated heavy metals are among the key factors that affect bacterial diversity and ecosystem functioning in freshwater systems.

Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach.

16S small-subunit (SSU) rRNA gene-based bacterial profiling is the gold standard for cost-effective taxonomic reconstruction of complex bacterial populations down to the genus level. However, it has been proven ineffective in clinical and research settings requiring higher taxonomic resolution. We therefore developed a bacterial profiling method based on the internal transcribed spacer (ITS) region employing optimized primers and a comprehensive ITS database for accurate cataloguing of bacterial communities at (sub)species resolution. Performance of the microbial ITS profiling pipeline was tested through analysis of host-associated, food, and environmental matrices, while its efficacy in clinical settings was assessed through analysis of mucosal biopsy specimens of colorectal cancer, leading to the identification of putative novel biomarkers. The data collected indicate that the proposed pipeline represents a major step forward in cost-effective identification and screening of microbial biomarkers at (sub)species level, with relevant impact in research, industrial, and clinical settings.IMPORTANCE We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings.

Soil microbial communities associated with giant sequoia: How does the world's largest tree affect some of the world's smallest organisms?

Giant sequoia (Sequoiadendron giganteum) is an iconic conifer that lives in relict populations on the western slopes of the California Sierra Nevada. In these settings, it is unusual among the dominant trees in that it associates with arbuscular mycorrhizal fungi rather than ectomycorrhizal fungi. However, it is unclear whether differences in microbial associations extend more broadly to nonmycorrhizal components of the soil microbial community. To address this question, we used next‐generation amplicon sequencing to characterize bacterial/archaeal and fungal microbiomes in bulk soil (0–5 cm) beneath giant sequoia and co‐occurring sugar pine (Pinus lambertiana) individuals. We did this across two groves with distinct parent material in Yosemite National Park, USA. We found tree‐associated differences were apparent despite a strong grove effect. Bacterial/archaeal richness was greater beneath giant sequoia than sugar pine, with a core community double the size. The tree species also harbored compositionally distinct fungal communities. This pattern depended on grove but was associated with a consistently elevated relative abundance of Hygrocybe species beneath giant sequoia. Compositional differences between host trees correlated with soil pH and soil moisture. We conclude that the effects of giant sequoia extend beyond mycorrhizal mutualists to include the broader community and that some but not all host tree differences are grove‐dependent.

PVAmpliconFinder: a workflow for the identification of human papillomaviruses from high-throughput amplicon sequencing

BackgroundThe detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods.ResultsWe have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals.ConclusionsPVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.

A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries.

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

The Composition and Function of Microbiomes Within Microcystis Colonies Are Significantly Different Than Native Bacterial Assemblages in Two North American Lakes.

The toxic cyanobacterium Microcystis is one of the most pervasive harmful algal bloom (HAB) genera and naturally occurs in large colonies known to harbor diverse heterotrophic bacterial assemblages. While colony-associated microbiomes may influence Microcystis blooms, there remains a limited understanding of the structure and functional potential of these communities and how they may be shaped by changing environmental conditions. To address this gap, we compared the dynamics of Microcystis-attached (MCA), free-living (FL), and whole water (W) microbiomes during Microcystis blooms using next-generation amplicon sequencing (16S rRNA), a predictive metagenome software, and other bioinformatic approaches. Microbiomes were monitored through high resolution spatial-temporal surveys across two North American lakes, Lake Erie (LE) and Lake Agawam (LA; Long Island, NY, United States) in 2017, providing the largest dataset of these fractions to date. Sequencing of 126 samples generated 7,922,628 sequences that clustered into 7,447 amplicon sequence variants (ASVs) with 100% sequence identity. Across lakes, the MCA microbiomes were significantly different than the FL and W fractions being significantly enriched in Gemmatimonadetes, Burkholderiaceae, Rhizobiales, and Cytophagales and depleted of Actinobacteria. Further, although MCA communities harbored > 900 unique ASVs, they were significantly less diverse than the other fractions with diversity inversely related to bloom intensity, suggesting increased selection pressure on microbial communities as blooms intensified. Despite taxonomic differences between lakes, predicted metagenomes revealed conserved functional potential among MCA microbiomes. MCA communities were significantly enriched in pathways involved in N and P cycling and microcystin-degradation. Taxa potentially capable of N2-fixation were significantly enriched (p < 0.05) and up to four-fold more abundant within the MCA faction relative to other fractions, potentially aiding in the proliferation of Microcystis blooms during low N conditions. The MCA predicted metagenomes were conserved over 8 months of seasonal changes in temperature and N availability despite strong temporal succession in microbiome composition. Collectively, these findings indicate that Microcystis colonies harbor a statistically distinct microbiome with a conserved functional potential that may help facilitate bloom persistence under environmentally unfavorable conditions.

The prognostic role of somatic BRCA mutations in ovarian cancer: Preliminary results from an observational multicenter cohort study.

e13674 Background: BRCA germline (gBRCA) mutations occur in 11-15% of women with unselected ovarian cancers (OC), whereas somatic BRCA (sBRCA) mutations occur in approximately 5-7% of cases. The impact of sBRCA mutations on OC outcome is still debated. Methods: With the aim to explore the prognostic role of sBRCA mutations, the BRCA mutational status of 149 non-mucinous and non-borderline OC and their clinical-pathological features were evaluated. BRCA1 and BRCA2 mutational profiles, either for sequence variants or copy number alterations, were evaluated by amplicon next-generation sequencing (NGS) technology. Results: 29 (19.5%) patients carried a gBRCA mutation (12.7% BRCA1 and 6.7% BRCA2). 26 (17.5%) patients presented a sBRCA mutation (6.7% BRCA1, 10% BRCA2, 0.6% BRCA1+BRCA2). Patients carrying a gBRCA mutation were slightly younger (57 years) than the others (60 years). The FIGO stage at the diagnosis was III-IV in 77.2% of cases, with no significant difference among subgroups. The most frequent histotype was serous for all the subgroups (93.1% in gBRCA, 84.6% in sBRCA, 77.7% in BRCA-negative, p = 0.08). 80.7% of sBRCA mutation carriers, 62.1% of gBRCA mutation carriers and 62.7% of BRCA-negative patients underwent upfront surgery (p = 0.46). 29.1% of sBRCA mutation carriers, 17.8% of gBRCA mutation carriers and 25.6% of BRCA-negative patients presented macroscopic residual disease after surgery (p = 0.68). Although non-statistically significant, gBRCA-associated OC were more likely to be platinum-sensitive (96.6%) than the other patients (92% in sBRCA and 87.1% in BRCA-negative patients). Overall, the median platinum-free interval (PFI) resulted shorter in sBRCA mutation patients compared to gBRCA and BRCA-negative patients (p = 0.051). No patient took PARP inhibitors as maintenance after the first line therapy. Also progression free survival 2 (PFS2) resulted shorter for sBRCA mutation patients (p = 0.008). Three sBRCA and 5 gBRCA mutation carriers took a PARP inhibitor as maintenance after the second line therapy. Finally, sBRCA mutated patients showed worse overall survival (OS) compared to the other subgroups (p = 0.014). Conclusions: Overall, 19.5% of OC patients presented a gBRCA mutation, while 17.5% of patients showed sBRCA mutations. sBRCA-related OC did not show significantly differences in clinical-pathological features (stage at diagnosis, histotype, time to surgery and residual after surgery). To our knowledge this is the first study showing shorter PFI, PFS2 and OS in patients carrying sBRCA mutations.

Are bacterial communities associated with microplastics influenced by marine habitats?

This study investigates the influence of marine habitats (i.e., the intertidal zone, supralittoral zone, and seawater), and polymer types (i.e., polyethylene, PE; polyethylene terephthalate, PET) on the diversity and structure of bacterial communities in marine microplastics. A three-month exposure experiment was conducted in Zhairuoshan Island, Zhoushan, China, a typical caldera volcanic island with minor anthropogenic disturbances. At the end of the exposure period, the transition for peaks corresponding to oxidized groups was observed using micro-Fourier transform infrared spectroscopy. Damages, including pits and cracks, and microorganisms were observed on the surfaces of the PE and PET pellets using scanning electron microscopy. Next-generation amplicon sequencing of the bacterial communities that had colonized the microplastics revealed that bacterial composition significantly varied depending on marine habitats and exposure times, rather than polymer type. Plastic debris in the intertidal zone exhibited the highest bacterial richness and diversity, and Bacillus was considered a potential degrader of plastic debris. The findings demonstrate that bacterial communities that colonize on microplastics are more potentially shaped by marine habitat and exposure time, and this would deepen our understanding of the ecological niche of microplastics surface.

Coastal urbanization influences human pathogens and microdebris contamination in seafood

Seafood is one of the leading imported products implicated in foodborne outbreaks worldwide. Coastal marine environments are being increasingly subjected to reduced water quality from urbanization and leading to contamination of important fishery species. Given the importance of seafood exchanged as a global protein source, it is imperative to maintain seafood safety worldwide. To illustrate the potential health risks associated with urbanization in a coastal environment, we use next-generation high-throughput amplicon sequencing of the 16S ribosomal RNA gene combined with infrared spectroscopy to characterize and quantify a vast range of potential human bacterial pathogens and microdebris contaminants in seawater, sediment and an important oyster fishery along the Mergui Archipelago in Myanmar. Through the quantification of >1.25 million high-quality bacterial operational taxonomic unit (OTU) reads, we detected 5459 potential human bacterial pathogens belonging to 87 species that are commonly associated with gut microbiota and an indication of terrestrial runoff of human and agricultural waste. Oyster tissues contained 51% of all sequenced bacterial pathogens that are considered to be both detrimental and of emerging concern to human health. Using infrared spectroscopy, we examined a total of 1225 individual microdebris particles, from which we detected 78 different types of contaminant materials. The predominant microdebris contaminants recovered from oyster tissues included polymers (48%), followed by non-native minerals (20%), oils (14%) and milk supplement powders (14%). Emerging technologies provide novel insights into the impacts of coastal development on food security and risks to human and environmental health.

Diet change affects intestinal microbiota restoration and improves vertical sleeve gastrectomy outcome in diet-induced obese rats

PurposeObesity, a worldwide health problem, is linked to an abnormal gut microbiota and is currently most effectively treated by bariatric surgery. Our aim was to characterize the microbiota of high-fat fed Sprague–Dawley rats when subjected to bariatric surgery (i.e., vertical sleeve gastrectomy) and posterior refeeding with either a high-fat or control diet. We hypothesized that bariatric surgery followed by the control diet was more effective in reverting the microbiota modifications caused by the high-fat diet when compared to either of the two factors alone.MethodsUsing next-generation sequencing of ribosomal RNA amplicons, we analyzed and compared the composition of the cecal microbiota after vertical sleeve gastrectomy with control groups representing non-operated rats, control fed, high-fat fed, and post-operative diet-switched animals. Rats were fed either a high-fat or control low-fat diet and were separated into three comparison groups after eight weeks comprising no surgery, sham surgery, and vertical sleeve gastrectomy. Half of the rats were then moved from the HFD to the control diet. Using next-generation sequencing of ribosomal RNA amplicons, we analyzed the composition of the cecal microbiota of rats allocated to the vertical sleeve gastrectomy group and compared it to that of the non-surgical, control fed, high-fat fed, and post-operative diet-switched groups. Additionally, we correlated different biological parameters with the genera exhibiting the highest variation in abundance between the groups.ResultsThe high-fat diet was the strongest driver of altered taxonomic composition, relative microbial abundance, and diversity in the cecum. These effects were partially reversed in the diet-switched cohort, especially when combined with sleeve gastrectomy, resulting in increased diversity and shifting relative abundances. Several highly-affected genera were correlated with obesity-related parameters.ConclusionsThe dysbiotic state caused by high-fat diet was improved by the change to the lower fat, higher fiber control diet. Bariatric surgery contributed significantly and additively to the diet in restoring microbiome diversity and complexity. These results highlight the importance of dietary intervention following bariatric surgery for improved restoration of cecal diversity, as neither surgery nor change of diet alone had the same effects as when combined.

Rapid, Inexpensive Measurement of Synthetic Bacterial Community Composition by Sanger Sequencing of Amplicon Mixtures.

SummarySynthetic bacterial communities are powerful tools for studying microbial ecology and evolution, as they enable rapid iteration between controlled laboratory experiments and theoretical modeling. However, their utility is hampered by the lack of fast, inexpensive, and accurate methods for quantifying bacterial community composition. Although next-generation amplicon sequencing can be very accurate, high costs (>$30 per sample) and turnaround times (>1 month) limit the nature and pace of experiments. Here, we quantify amplicon composition in synthetic bacterial communities through Sanger sequencing. We PCR amplify a universal marker gene, then we sequence this amplicon mixture in a single Sanger sequencing reaction. We then fit the “mixed” electropherogram with contributions from each community member as a linear combination of time-warped single-strain electropherograms, allowing us to estimate the fractional amplicon abundance of each strain within the community. This approach can provide results within one day and costs ∼$5 per sample.

Abstract P6-08-13: Personalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing

Abstract Background: Germline copy number variants (CNV) of hereditary breast and ovarian cancer genes are a known class of clinically significant mutations. However, failure to detect CNV is a known limitation of Sanger sequencing and conventional amplicon next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA), while robust for CNV detection, is labor-intensive, costly to scale up and has limited breakpoint resolution at exon level only. We evaluated the clinical utility of molecular-barcode NGS and long-read nanopore sequencing in our genetic testing algorithm, specifically on CNV detection and breakpoint characterization of probands and family members. Methods: Both molecular-barcode NGS (BRCA1, BRCA2, TP53, PTEN, PALB2 and CDH1) and MLPA (BRCA1 and BRCA2) were performed in parallel on 200 high-risk breast and/or ovarian cancer probands and 11 CNV positive controls. CNV calling from MLPA and NGS data was performed by Coffalyser.Net and a novel in-house bioinformatics algorithm BAMClipper-SE, respectively. Nanopore amplicon or whole-genome sequencing was attempted to map the exact breakpoint of CNV controls and any newly identified CNV positive probands (up to May 2019). Personalized breakpoint PCR test for specific CNVs was designed and validated on probands and subsequently applied to corresponding family members for carrier testing. Results: Molecular-barcode NGS was 100% sensitive in detecting all BRCA1/2 CNVs from 11 positive controls. A total of 4 BRCA1/2 CNVs were detected by NGS from the 200 probands and matched the corresponding MLPA results. In addition, a PALB2 CNV (exons 4-6 deletion) was also detected by NGS during the parallel comparison. Up to May 2019, a total of 22 probands with detectable CNV in BRCA1, BRCA2, PALB2 or CHEK2 were accrued. Exact CNV breakpoint at nucleotide resolution was identified from 20 probands by nanopore and Sanger sequencing (BRCA1 n=12, BRCA2 n=5, PALB2 n=2 and CHEK2 n=1). High-resolution breakpoint mapping was applicable to inform variant pathogenicity and to enable recurrent CNV identification. Personalized breakpoint PCR designed for the 20 probands were applied to 42 family members, of whom 16 were CNV carriers and 26 were non-carriers. Breakpoint PCR results and any available MLPA results of these family members were 100% concordant. Conclusion: Molecular-barcode NGS allows a streamlined workflow to detect both sequence mutations and CNVs. It proves to be a reliable and cost-effective replacement of BRCA1/2 MLPA in genetic testing algorithm of new probands. Simultaneously the NGS workflow expands CNV detection coverage to additional genes (e.g. PALB2) without costly scale-up of MLPA gene panels. Nanopore sequencing is a practical alternative to MLPA in orthogonal validation of newly detected CNV. The high-resolution breakpoint mapping transforms CNV carrier testing to a personalized, simple and cost-effective breakpoint PCR. Citation Format: Ava Kwong, Chun H Au, Dona N Ho, Elaine YL Wong, Yvonne Chung, Fian BF Law, Cecilia YS Ho, Jiawei Chen, Isabella W Cheuk, Vivian Y Shin, Tsun L Chan, Hextan YS Ngan, Edmond SK Ma. Personalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-13.