scholarly journals A Round Trip to the Desert: In situ Nanopore Sequencing Informs Targeted Bioprospecting

2021 ◽  
Vol 12 ◽  
Author(s):  
Adriel Latorre-Pérez ◽  
Helena Gimeno-Valero ◽  
Kristie Tanner ◽  
Javier Pascual ◽  
Cristina Vilanova ◽  
...  
Keyword(s):  
16S Rrna Gene Sequencing ◽  
Culture Collection ◽  
Rrna Gene ◽  
Culturable Bacteria ◽  
Nanopore Sequencing ◽  
Mobile Laboratory ◽  
Round Trip ◽  
Laboratory Results ◽  
Rrna Gene Sequencing

Bioprospecting expeditions are often performed in remote locations, in order to access previously unexplored samples. Nevertheless, the actual potential of those samples is only assessed once scientists are back in the laboratory, where a time-consuming screening must take place. This work evaluates the suitability of using Nanopore sequencing during a journey to the Tabernas Desert (Spain) for forecasting the potential of specific samples in terms of bacterial diversity and prevalence of radiation- and desiccation-resistant taxa, which were the target of the bioprospecting activities. Samples collected during the first day were analyzed through 16S rRNA gene sequencing using a mobile laboratory. Results enabled the identification of locations showing the greatest and the least potential, and a second, informed sampling was performed focusing on those sites. After finishing the expedition, a culture collection of 166 strains belonging to 50 different genera was established. Overall, Nanopore and culturing data correlated well, since samples holding a greater potential at the microbiome level also yielded a more interesting set of microbial isolates, whereas samples showing less biodiversity resulted in a reduced (and redundant) set of culturable bacteria. Thus, we anticipate that portable sequencers hold potential as key, easy-to-use tools for in situ-informed bioprospecting strategies.

scholarly journals Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1105 ◽  
Author(s):  
Astrid P. Heikema ◽  
Deborah Horst-Kreft ◽  
Stefan A. Boers ◽  
Rick Jansen ◽  
Saskia D. Hiltemann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Rrna Gene Sequencing ◽  
Oxford Nanopore Technologies ◽  
Nasal Microbiota

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies—ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies—ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies—ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.

scholarly journals Beyond Archaea: The Table Salt Bacteriome

2021 ◽  
Vol 12 ◽  
Author(s):  
Leila Satari ◽  
Alba Guillén ◽  
Adriel Latorre-Pérez ◽  
Manuel Porcar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Halophilic Bacteria ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Culturable Bacteria ◽  
Table Salt ◽  
Culture Independent ◽  
Rrna Gene Sequencing ◽  
Marine Salt

Commercial table salt is a condiment with food preservative properties by decreasing water activity and increasing osmotic pressure. Salt is also a source of halophilic bacteria and archaea. In the present research, the diversity of halotolerant and halophilic microorganisms was studied in six commercial table salts by culture-dependent and culture-independent techniques. Three table salts were obtained from marine origins: Atlantic Ocean, Mediterranean (Ibiza Island), and Odiel marshes (supermarket marine salt). Other salts supplemented with mineral and nutritional ingredients were also used: Himalayan pink, Hawaiian black, and one with dried vegetables known as Viking salt. The results of 16S rRNA gene sequencing reveal that the salts from marine origins display a similar archaeal taxonomy, but with significant variations among genera. Archaeal taxa Halorubrum, Halobacterium, Hallobellus, Natronomonas, Haloplanus, Halonotius, Halomarina, and Haloarcula were prevalent in those three marine salts. Furthermore, the most abundant archaeal genera present in all salts were Natronomonas, Halolamina, Halonotius, Halapricum, Halobacterium, Haloarcula, and uncultured Halobacterales. Sulfitobacter sp. was the most frequent bacteria, represented almost in all salts. Other genera such as Bacillus, Enterococcus, and Flavobacterium were the most frequent taxa in the Viking, Himalayan pink, and black salts, respectively. Interestingly, the genus Salinibacter was detected only in marine-originated salts. A collection of 76 halotolerant and halophilic bacterial and haloarchaeal species was set by culturing on different media with a broad range of salinity and nutrient composition. Comparing the results of 16S rRNA gene metataxonomic and culturomics revealed that culturable bacteria Acinetobacter, Aquibacillus, Bacillus, Brevundimonas, Fictibacillus, Gracilibacillus, Halobacillus, Micrococcus, Oceanobacillus, Salibacterium, Salinibacter, Terribacillus, Thalassobacillus, and also Archaea Haloarcula, Halobacterium, and Halorubrum were identified at least in one sample by both methods. Our results show that salts from marine origins are dominated by Archaea, whereas salts from other sources or salt supplemented with ingredients are dominated by bacteria.

Detection of WWE2-relatedLentisphaeraeby 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

2010 ◽  
Vol 56 (10) ◽  
pp. 846-852 ◽  
Author(s):  
Rim Driss Limam ◽  
Théodore Bouchez ◽  
Rakia Chouari ◽  
Tianlun Li ◽  
Insaf Barkallah ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Fluorescence In Situ Hybridization ◽  
Landfill Leachate ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Victivallis Vadensis

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.

scholarly journals Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

2016 ◽  
Vol 113 (28) ◽  
pp. E4069-E4078 ◽  
Author(s):  
Roland Hatzenpichler ◽  
Stephanie A. Connon ◽  
Danielle Goudeau ◽  
Rex R. Malmstrom ◽  
Tanja Woyke ◽  
...  
Keyword(s):  
16S Rrna Gene Sequencing ◽  
Microbial Consortia ◽  
Rrna Gene ◽  
Anaerobic Oxidation Of Methane ◽  
Bacterial Consortia ◽  
Symbiotic Relationships ◽  
Oxidation Of Methane ◽  
Translational Activity ◽  
Rrna Gene Sequencing

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

scholarly journals Localization and Visualization of a Coxiella-Type Symbiont within the Lone Star Tick, Amblyomma americanum

2007 ◽  
Vol 73 (20) ◽  
pp. 6584-6594 ◽  
Author(s):  
Olga Klyachko ◽  
Barry D. Stein ◽  
Nathan Grindle ◽  
Keith Clay ◽  
Clay Fuqua
Keyword(s):  
16S Rrna Gene Sequencing ◽  
Amblyomma Americanum ◽  
Malpighian Tubules ◽  
Rrna Gene ◽  
Wide Field ◽  
Fluorescent Staining ◽  
Diagnostic Pcr ◽  
Transmission Electron ◽  
Rrna Gene Sequencing

ABSTRACT A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.

scholarly journals 72 PRELIMINARY DATA ON THE PRESENCE OF BACTERIA IN THE UTERUS OF PREGNANT COWS

2016 ◽  
Vol 28 (2) ◽  
pp. 165
Author(s):  
H. G. Pedersen ◽  
L. R. V. Knudsen ◽  
J. S. Agerholm ◽  
T. K. Jensen ◽  
K. S. Klitgaard ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Bacterial Invasion ◽  
Rrna Gene ◽  
Epithelial Surface ◽  
Pregnant Uterus ◽  
Rrna Gene Sequencing

Bacterial invasion of the uterus during the postpartum period has been well described. Recent papers using 16S rRNA gene sequencing techniques suggest that the nonpregnant uterus contains a diverse flora of bacteria that are not necessarily pathogenic. In contrast, the pregnant uterus has until now been considered a sterile environment. The aim of the present study was to investigate whether bacteria were present in the uteri of pregnant cows. Uteri from pregnant, slaughtered animals (n = 47) were sampled. The surface of the uterus was wiped with alcohol, flame sterilized, and cut open with sterile scissors. Samples were taken from the endometrium and from the placentomes. The samples were embedded in paraffin, sectioned at 3 microns, and prepared for fluorescence in situ hybridization using a probe targeting the 16S rRNA of the domain bacteria, so that all bacteria regardless of species were visualised. Using fluorescence microscopy, the presence of bacteria within or on the surface of the endometrium and within the placentomes was noted. The stage of pregnancy was estimated to range from 26 to 263 days by measuring the size of the embryo or fetus. The endometrial samples from 85.1% (40/47) of pregnant cows contained bacteria. In 22 cows, the bacteria were localised within the endometrial tissue, whereas in the remaining 18 cows, the bacteria were on the epithelial surface. Placental samples were obtained from 43 cows, and 76.7% (33/43) of these contained bacteria. The presence of bacteria in the pregnant uterus may suggest that a cow can carry a pregnancy despite the presence of few potentially pathogenic bacteria or that normal flora exist in the uterus as in, for example, the vagina. In conclusion, bacteria were present in the endometrium and placentomes of pregnant cows. Further analyses using rRNA gene sequencing techniques will aim to confirm the presence of bacteria in the bovine pregnant uterus and to investigate which species of bacteria are present in the uterus during pregnancy.

scholarly journals Comparison of Illumina Versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

2020 ◽  
Author(s):  
Astrid. P. Heikema ◽  
Deborah Horst-Kreft ◽  
Stefan A. Boers ◽  
Rick Jansen ◽  
Saskia D. Hiltemann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Single Species ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Rrna Gene Sequencing

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME and an in house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 and R9.2 were compared.

scholarly journals Pantoea rodasii sp. nov., Pantoea rwandensis sp. nov. and Pantoea wallisii sp. nov., isolated from Eucalyptus

2012 ◽  
Vol 62 (Pt_7) ◽  
pp. 1457-1464 ◽  
Author(s):  
Carrie L. Brady ◽  
Ilse Cleenwerck ◽  
Lorinda van der Westhuizen ◽  
Stephanus N. Venter ◽  
Teresa A. Coutinho ◽  
...  
Keyword(s):  
South Africa ◽  
Type Strain ◽  
Type Species ◽  
Novel Species ◽  
16S Rrna Gene Sequencing ◽  
Culture Collection ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Rrna Gene Sequencing

Several Gram-negative-staining, facultatively anaerobic bacterial isolates were obtained from Eucalyptus seedlings showing symptoms of bacterial blight and dieback in Colombia, Rwanda and South Africa. Partial 16S rRNA gene sequencing, together with partial gyrB sequencing, placed the isolates in the genus Pantoea and indicated that they constituted three novel species. Multilocus sequence analysis (MLSA) based on partial sequences of gyrB, rpoB, infB and atpD revealed Pantoea dispersa , Pantoea eucrina and Pantoea cypripedii as their closest phylogenetic relatives. DNA–DNA hybridization studies confirmed the classification of the new isolates as three novel species and phenotypic tests allowed them to be differentiated from their closest phylogenetic neighbours. The names Pantoea rodasii sp. nov. [type strain LMG 26273T = BD 943T (deposited with the Plant Pathogenic and Plant Protecting Bacteria Collection, South Africa) = BCC 581T (deposited with the Bacterial Culture Collection, Forestry and Agricultural Institute, South Africa)], Pantoea rwandensis sp. nov. (type strain LMG 26275T = BD 944T = BCC 571T) and Pantoea wallisii sp. nov. (type strain LMG 26277T = BD 946T = BCC 682T) are proposed.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

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