Amplicon Sequencing
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2021 ◽  
Anders Kiledal ◽  
Julia A Maresca

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

2021 ◽  
Vol 12 ◽  
Yuanhui Liu ◽  
Bin Ma ◽  
Wenfeng Chen ◽  
Klaus Schlaeppi ◽  
Matthias Erb ◽  

Root-microbiome interactions are of central importance for plant performance and yield. A distinctive feature of legumes is that they engage in symbiosis with N2-fixing rhizobia. If and how the rhizobial symbiotic capacity modulates root-associated microbiomes are still not yet well understood. We determined root-associated microbiomes of soybean inoculated with wild type (WT) or a noeI mutant of Bradyrhizobium diazoefficiens USDA 110 by amplicon sequencing. UPLC-MS/MS was used to analyze root exudates. The noeI gene is responsible for fucose-methylation of Nod factor secreted by USDA 110 WT strain. Soybean roots inoculated with the noeI mutant showed a significant decrease in nodulation and root-flavonoid exudation compared to roots inoculated with WT strain. The noeI mutant-inoculated roots exhibited strong changes in microbiome assembly in the rhizosphere and rhizoplane, including reduced diversity, changed co-occurrence interactions and a substantial depletion of root microbes. Root exudates and soil physiochemical properties were significantly correlated with microbial community shift in the rhizosphere between different rhizobial treatments. These results illustrate that rhizobial symbiotic capacity dramatically alters root-associated microbiomes, in which root exudation and edaphic patterns play a vital role. This study has important implications for understanding the evolution of plant-microbiome interactions.

Zoonoses ◽  
2021 ◽  
Vol 1 (1) ◽  
Kai Song ◽  
Yuxin Ji ◽  
Surong Sun ◽  
Xihong Yue ◽  
Cheng Wang ◽  

Background: Ticks are a major arthropod vector of zoonotic diseases affecting both humans and domestic animals worldwide. Thus, studying tick microbiota would aid in understanding of the potential threats posed by ticks. Methods: Approximately 8,000 unfed ticks, identified as Dermacentor nuttalli, were collected from the sylvosteppe in the western Tianshan mountains. To investigate their potential pathogens, we divided the ticks into 36 groups of 200–300 individuals each for examination with culturomics and 16S rDNA amplicon sequencing. Results: A total of 237 bacterial genera were identified with the two methods. Culturomics identified 46 bacterial species from 23 genera, predominantly Pseudomonas, Pantoea, and Bacillus, whereas 16S rDNA sequencing identified 461 OTUs from 233 genera, predominantly Pseudomonas (53.8%), Coxiella (17.2%), and Pantoea (6.4%). Coxiella, Rickettsia, and ten other genera were discovered only by sequencing, because optimal cultivating conditions were not used for their isolation, whereas Arthrobacter and three other genera were discovered only through culturomics. Conclusions: Several of the identified bacteria, such as line-related sepsis-causing Delftia acidovorans and the pneumonia agent Acinetobacter pittii, can cause human diseases. Thus, both sequencing and culturomics methods are crucial for comprehensive understanding of the microbiota of D. nuttalli.

2021 ◽  
Vol 12 ◽  
Nicole E. Adams ◽  
Madeleine A. Becker ◽  
Suzanne Edmands

With developing understanding that host-associated microbiota play significant roles in individual health and fitness, taking an interdisciplinary approach combining microbiome research with conservation science is increasingly favored. Here we establish the scat microbiome of the imperiled Channel Island fox (Urocyon littoralis) and examine the effects of geography and captivity on the variation in bacterial communities. Using high throughput 16S rRNA gene amplicon sequencing, we discovered distinct bacterial communities in each island fox subspecies. Weight, timing of the sample collection, and sex contributed to the geographic patterns. We uncovered significant taxonomic differences and an overall decrease in bacterial diversity in captive versus wild foxes. Understanding the drivers of microbial variation in this system provides a valuable lens through which to evaluate the health and conservation of these genetically depauperate foxes. The island-specific bacterial community baselines established in this study can make monitoring island fox health easier and understanding the implications of inter-island translocation clearer. The decrease in bacterial diversity within captive foxes could lead to losses in the functional services normally provided by commensal microbes and suggests that zoos and captive breeding programs would benefit from maintaining microbial diversity.

2021 ◽  
Vol 8 ◽  
Chuya Shinzato ◽  
Haruhi Narisoko ◽  
Koki Nishitsuji ◽  
Tomofumi Nagata ◽  
Noriyuki Satoh ◽  

Coral reefs, the most biodiverse habitats in the ocean, are formed by anthozoan cnidarians, the scleractinian corals. Recently, however, ongoing climate change has imperiled scleractinian corals and coral reef environments are changing drastically. Thus, convenient, high-density monitoring of scleractinian corals is essential to understand changes in coral reef communities. Environmental DNA (eDNA) metabarcoding is potentially one of the most effective means of achieving it. Using publicly available scleractinian mitochondrial genomes, we developed high-specificity primers to amplify mitochondrial 12S ribosomal RNA (12S) and cytochrome oxidase-1 (CO1) genes of diverse scleractinian corals, which could be used for genus-level metabarcoding analyses, using next-generation sequencing technologies. To confirm the effectiveness of these primers, PCR amplicon sequencing was performed using eDNA isolated along the seashore of Okinawa, Japan. We successfully amplified all eDNA samples using PCR. Approximately 93 and 72% of PCR amplicon sequences of 12S and CO1 primers originated from scleractinian 12S and CO1 genes, respectively, confirming higher specificities for coral mitochondrial genes than primers previously used for coral eDNA metabarcoding. We also found that hierarchical clustering, based on the percentage of mapped reads to each scleractinian genus, discriminates between sampling locations, suggesting that eDNA surveys are sufficiently powerful to reveal differences between coral communities separated by <1 km. We conclude that the method reported here is a powerful tool for conducting efficient eDNA surveys targeting scleractinian corals.

2021 ◽  
Vol 232 (12) ◽  
István Szabó ◽  
Jafar Al-Omari ◽  
Gábor Soma Szerdahelyi ◽  
Milán Farkas ◽  
Yazid Al-Omari ◽  

AbstractDespite the great benefits of plastics in different aspects of life and due to the increase in plastic production and use, plastic wastes are becoming a major environmental concern. It is well known that inappropriate use and disposal lead to the accumulation of plastic litter in different aquatic environments. Microbial biofilm is able to develop on the surface of plastics (plastisphere) in aquatic environments over time. The aim of this study was to describe the bacterial communities associated with plastics in freshwater. Thus, in our first test, a total of six self-designed plastic colonizers were submerged under the surface of the water in Vácszentlászló lake, located in central Hungary, for a period of 3 months. Two plastic colonizers were cultivated monthly. Associated microbial communities were then analyzed as follows: (a) bacterial communities were studied by amplicon sequencing and (b) culturable bacteria were isolated from plastic surfaces and identified by 16S rRNA gene sequencing. Coinciding with these analyses of plastic colonizing communities, surface water samples from the lake were also taken, and in a second test, other materials (eg. wood, glass) associated bacterial communities were also investigated with the same methods. Amplicon sequencing showed notable differences between the plastic and other materials colonizing, and lake waterborne microbial community composition. Using the LB agar, no novel species were found; however, several known pathogenic species were identified. The self-designed plastic colonizer was successfully used during the winter over a 3-month period, suggesting that it could be an appropriate method of choice to study microplastic-associated microbes for longer periods and in variable environmental conditions.

2021 ◽  
Vol 10 (15) ◽  
pp. e399101522919
Elizabeth Maria Bispo Beltrão ◽  
Érica Maria Oliveira ◽  
Crhisllane Rafaele dos Santos Vasconcelos Vasconcelos ◽  
Antônio Mauro Rezende ◽  
Ana Catarina de Souza Lopes

The present study objective to characterize the clinical aspects of a patient infected with two strains of P. mirabilis and the presence of resistance determinants in the two isolates from a patient at a public hospital in Recife-PE, Brazil. The total DNA of the isolates was extracted and submitted to PCR and amplicon sequencing for the investigation of resistance genes, blaKPC, blaOXA-10, blaOXA-23, blaOXA-48, blaOXA-58, blaVIM, blaIMP, blaSPM, blaGES, blaNDM, qnrD and aac(6')-Ib). Isolate P21-A2 harbored the aac(6')-Ib, blaOXA-10 and qnrD genes. One of the isolates, P20-A2, was selected for plasmid DNA sequencing. The results showed that the patient developed multiple infections with various pathogens including two strains of P. mirabilis. The patient was hospitalized for 103 days, had septic shock of skin, abdominal, pulmonary and ulcer focus, and died. Isolate P20-A2 harbored the genes blaNDM, qnrD, aph(3')-VI, blaKPC and blaOXA-10, and plasmids IncQ and Col3M, together with NTEKPC-IId. To our knowledge, this is the first report of P. mirabilis harboring NTEKPC-IId. Although P. mirabilis is standing out as a cause of nosocomial infections and a resistant multidrug pathogen, this species is still neglected, the emergence of these P. mirabilis isolates harboring aforementioned resistance determinants and the plasmids IncQ and Col3M demonstrate the potential for dissemination of important resistance genes, mainly in the case of P. mirabilis.

2021 ◽  
Vol 9 (12) ◽  
pp. 2445
Huatao Yuan ◽  
Tangcheng Li ◽  
Hongfei Li ◽  
Cong Wang ◽  
Ling Li ◽  

Free-living (FL) and particle-associated (PA) bacterioplankton communities play critical roles in biogeochemical cycles in the ocean. However, their community composition, assembly process and functions in the continental shelf and slope regions are poorly understood. Based on 16S rRNA gene amplicon sequencing, we investigated bacterial communities’ driving factors, assembly processes and functional potentials at a subtropical marginal sea. The bacterioplankton community showed specific distribution patterns with respect to lifestyle (free living vs. particle associated), habitat (slope vs. shelf) and depth (surface vs. DCM and Bottom). Salinity and water temperature were the key factors modulating turnover in the FL community, whereas nitrite, silicate and phosphate were the key factors for the PA community. Model analyses revealed that stochastic processes outweighed deterministic processes and had stronger influences on PA than FL. Homogeneous selection (Hos) was more responsible for the assembly and turnover of FL, while drift and dispersal limitation contributed more to the assembly of PA. Importantly, the primary contributor to Hos in PA was Gammaproteobacteria:Others, whereas that in FL was Cyanobacteria:Bin6. Finally, the PICRUSt2 analysis indicated that the potential metabolisms of carbohydrates, cofactors, amino acids, terpenoids, polyketides, lipids and antibiotic resistance were markedly enriched in PA than FL.

2021 ◽  
Jennifer J Dawkins ◽  
Jessica R Allegretti ◽  
Travis E Gibson ◽  
Emma McClure ◽  
Mary Delaney ◽  

Background Clostridioides difficile infection (CDI) is the most common hospital acquired infection in the U.S., with recurrence rates >15%. Although primary CDI has been extensively linked to gut microbial dysbiosis, less is known about the factors that promote or mitigate recurrence. Moreover, previous studies have not shown that microbial abundances in the gut measured by 16S rRNA amplicon sequencing alone can accurately predict CDI recurrence. Results We conducted a prospective, longitudinal study of 53 non-immunocompromised participants with primary CDI. Stool sample collection began pre-CDI antibiotic treatment at the time of diagnosis, and continued up to eight weeks post-antibiotic treatment, with weekly or twice weekly collections. Samples were analyzed using: (1) 16S rRNA amplicon sequencing, (2) liquid chromatography/mass-spectrometry metabolomics measuring 1387 annotated metabolites, and (3) short-chain fatty acid profiling. The amplicon sequencing data showed significantly delayed recovery of microbial diversity in recurrent participants, and depletion of key anaerobic taxa at multiple time-points, including Clostridium cluster XIVa and IV taxa. The metabolomic data also showed delayed recovery in recurrent participants, and moreover mapped to pathways suggesting distinct functional abnormalities in the microbiome or host, such as decreased microbial deconjugation activity, lowered levels of endocannabinoids, and elevated markers of host cell damage. Further, using predictive statistical/machine learning models, we demonstrated that the metabolomic data, but not the other data sources, can accurately predict future recurrence at one week (AUC 0.77 [0.71, 0.86; 95% interval]) and two weeks (AUC 0.77 [0.69, 0.85; 95% interval]) post-treatment for primary CDI. Conclusions The prospective, longitudinal and multi-omic nature of our CDI recurrence study allowed us to uncover previously unrecognized dynamics in the microbiome and host presaging recurrence, and, in particular, to elucidate changes in the understudied gut metabolome. Moreover, we demonstrated that a small set of metabolites can accurately predict future recurrence. Our findings have implications for development of diagnostic tests and treatments that could ultimately short-circuit the cycle of CDI recurrence, by providing candidate metabolic biomarkers for diagnostics development, as well as offering insights into the complex microbial and metabolic alterations that are protective or permissive for recurrence.

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Kevin Xu Zhong ◽  
Anna Cho ◽  
Christoph M. Deeg ◽  
Amy M. Chan ◽  
Curtis A. Suttle

Abstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. Results To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. Conclusion CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences.

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