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16S rRNA gene sequence analysis using the MinION™ nanopore sequencer. a Workflow of 16S rRNA gene amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Even with the full-length 16S rRNA gene analysis, species-level resolution is not possible for Bacillus and Escherichia. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the given level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown
Source publication
Background
Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S...
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Citations
... The introduction of long-read sequencing in 16S rRNA gene-targeted metagenomic studies has revolutionised the ability to sequence the entire 16S rRNA gene, overcoming the constraints of short-read technologies, such as Illumina, that were limited to hypervariable regions [3][4][5][6][7][8][9]. Pioneered by PacBio and Oxford Nanopore Technologies (ONT), long-read technologies offer the benefits of sequencing long stretches of DNA in a relatively high-throughput, culture-independent, manner. ...
Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND ( G enome-derived R ibosomal O pero n D atabase), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.
... Since the first study published in 2009 [1], the use of Next Generation Sequencing to analyze libraries of amplicons of taxonomically relevant regions of the genomes of Bacteria and Archaea (typically the 16S RNA gene [2][3][4]) and Fungi (typically one or more regions including the Internal Transcribed Spacer [5][6][7]) has become the standard tool for the cultivation independent analysis of microbial food communities [8][9][10][11]. The approaches used for sequencing, for the bioinformatic analysis of sequences and for the statistical analysis of the results have been reviewed multiple times [2,[12][13][14][15] and the increasing use of third generation platforms is helping in overcoming some of the limitations of short-read sequencing [16][17][18][19][20][21]. Raw sequences are often but not always made available in public repositories, like, among others, the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra, last accessed on 18 April 2024), the European Nucleotide Archive (ENA, https://www.ebi.ac.uk/ena/browser/ home, last accessed on 18 April 2024). ...
Amplicon-targeted metagenomics is now the standard approach for the study of the composition and dynamics of food microbial communities. Hundreds of papers on this subject have been published in scientific journals and the information is dispersed in a variety of sources, while raw sequences and their metadata are available in public repositories for some, but not all, of the published studies. A limited number of web resources and databases allow scientists to access this wealth of information but their level of annotation on studies and samples varies. Here, we report on the release of FoodMicrobionet v5, a comprehensive database of metataxonomic studies on bacterial and fungal communities of foods. The current version of the database includes 251 published studies (11 focusing on fungal microbiota, 230 on bacterial microbiota, and 10 providing data for both bacterial and fungal microbiota) and 14,035 samples with data on bacteria and 1114 samples with data on fungi. The new structure of the database is compatible with interactive apps and scripts developed for previous versions and allows scientists, R&D personnel in industries and regulators to access a wealth of information on food microbial communities.
... However, the sequencing platform allows only short-reads with a sequence length of <500 bp, restricting the coverage of the 16S gene to a maximum of two variable regions and limiting the taxonomic classication up to the genus level. [28][29][30] Third-generation sequencing, commonly referred to the sequencing platforms offered by Oxford Nanopore Sequencing (ONT) and Pacic Biosciences (PacBio), overcomes some of the major limitations of NGS by enabling long-read sequencing. 31,32 Among these techniques, MinION nanopore sequencing from ONT utilizes a protein nanopore complex to guide a DNA strand to translocate through the pore and determines the sequence from the changes in ionic conductivities as different nucleotide bases pass through the pore. ...
... [33][34][35][36][37] The long-read capability of nanopore sequencing allows for full-length 16S gene amplicon sequencing with the ability to discriminate up to the species level in a sample of mixed bacterial composition. 30 Furthermore, multiplexing the samples by barcoding enables running multiple samples on a single run, enhancing the throughput and reducing the cost. Together, these features of nanopore sequencing make it a potentially attractive procedure for the identication of bacterial colonies through 16S amplicon analysis. ...
... 20 However 16S taxonomical classication by Illumina-based sequencing usually targets the hypervariable regions V4, V3-V4, or V4-V5 of the 16S gene due to the limitation of this technique to read only a short span of the 16S sequence. 29,30 Such a restriction imposed on the amplicon length allows identication only up to the genus level. While near full-length 16S sequencing on the Illumina platform has been achieved by using unique, random sequences to tag individual 16S gene templates, the long, complex procedure is not practical for routine implementation. ...
Bioaerosol samples are characterized by very low biomass, so culture-based detection remains a reliable and acceptable technique to identify and quantify microbes present in these samples. The process typically involves the generation of bacterial colonies by inoculating the sample on an agar plate, followed by the identification of colonies through DNA sequencing of a PCR-amplified targeted gene. The Sanger method is often the default choice for sequencing, but its application might be limited in identifying multi-species microbial colonies that could potentially form from bacterial aggregates present in bioaerosols. In this work, we compared Sanger and MinION nanopore sequencing techniques in identifying bioaerosol-derived bacterial colonies using 16S rRNA gene analysis. We found that for five out of the seven colonies examined, both techniques indicated the presence of the same bacterial genus. For one of the remaining colonies, a noisy Sanger electropherogram failed to generate a meaningful sequence, but nanopore sequencing identified it to be a mix of two bacterial genera. For the other remaining colony, the Sanger sequencing suggested a single genus with a high sequence alignment and clean electropherogram; however, the nanopore sequencing suggested the presence of a second less abundant genus. These findings were further corroborated using mock colonies, where nanopore sequencing was found to be a superior method in accurately classifying individual bacterial components in mock multispecies colonies. Our results show the advantage of using nanopore sequencing over the Sanger method for culture-based analysis of bioaerosol samples, where direct inoculation to a culture plate could lead to the formation of multispecies colonies.
... In case of nanopore sequencing, lack of a DNA synthesis step during the sequencing step improves amplification bias for species abundance but does not remove it completely (Fig. 3) (Huggins et al. 2022). Application of optimized primers set for target barcode amplification can drastically improves PCR bias, as well as new possible selection and amplification strategies to create barcode libraries (Matsuo et al. 2021). ...
Although established biotechnological applications of microalgae e.g., the production of high-value metabolites is based on axenic cultures, exploitation of the mutualistic consortia of microalgae and bacteria quickly comes to foreground, especially in bioremediation and wastewater treatment. This trend shifts the focus from genomic research of certain microalgal species to metagenomic studies of interactions between microalgae and bacteria in natural communities and in artificial consortia. Dissection of the genetic determinants of the robustness and productivity of the consortia become a hot research direction, too. Admirable contribution to this topic had been made by high-throughput sequencing (HTS), while recent breakthrough in this field was entailed by the advent and rapid development of the 3rd generation nanopore sequencing which becomes increasingly accurate while providing unprecedented sequencing performance. Recent progress of the Oxford Nanopore Technologies (ONT) enabled both classical metagenomic analysis of microalgal-bacterial communities based on whole metagenome sequencing as well as taxonomic and genetic profiling based on the amplicon sequencing. The parallel emergence of novel bioinformatic algorithms for processing the metagenomic datasets opened new opportunities for the analysis of structure and physiology of microalgal-bacterial communities. From the practical perspective, the new HTS techniques became a time- and labor-savers in discovery of new microalgae with a high potential for the accumulation of valuable metabolites, biodegradation of hazardous micropollutants, and biosequestration of nutrients from waste streams. Search for prokaryotic species boosting the biotechnological potential of eukaryotic microalgae via mutualistic interactions with them is another important goal. The insights from the both short-read and long-read metagenomics will form a solid foundation for the rational design of microalgal-bacterial consortia for biotechnology. In this review, we briefly outline the benefits of the long-read sequencing for structural and functional investigation of algal-bacterial consortia and summarize recent reports on using this approach for achieving the biotechnology-related goals.
... Analyzing the changes in composition and abundance of microbial communities provides valuable insight into ecosystem dynamics, including health, stability, and resilient changes, along with the ability to identify biomarkers that can serve as indicators for monitoring environmental changes, treatment responses, or disease [1]. Targeted sequencing is typically conducted based on conserved regions containing phylogenetically informative polymorphisms, such as the 16S rRNA gene for prokaryotes and the 18S rRNA or ITS genes for eukaryotes, which offer a powerful and economical way to characterize the bacterial community in large numbers of samples with affordable techniques [2][3][4]. The microbiome analysis pipeline includes mainly the clustering of sequences into OTUs or ASVs (Operational Taxonomic Units or Amplicon Sequence Variants) followed by taxonomic classification of the representative sequences. ...
Over the last decades, the analysis of complex microbial communities by high-throughput sequencing of marker gene amplicons has become routine work for many research groups. However, the main challenges faced by scientists who want to make use of the generated sequencing datasets are the lack of expertise to select a suitable pipeline and the need for bioinformatics or programming skills to apply it. Here, we present MetaXplore, an interactive, user-friendly platform that enables the discovery and visualization of amplicon sequencing data. Currently, it provides a set of well-documented choices for downstream analysis, including alpha and beta diversity analysis, taxonomic composition, differential abundance analysis, identification of the core microbiome within a population, and biomarker analysis. These features are presented in a user-friendly format that facilitates easy customization and the generation of publication-quality graphics. MetaXplore is implemented entirely in the R language using the Shiny framework. It can be easily used locally on any system with R installed, including Windows, Mac OS, and most Linux distributions, or remotely via a web server without bioinformatic expertise. It can also be used as a framework for advanced users who can modify and expand the tool.
... This sequencing platform offers a culture-free method that both provides a cost-effective technique and is associated with a number of essential benefits regarding long-read data (26). The amplification and sequencing of the full-length 16S rDNA gene (~1,500 bp) can allow bacterial identification up to the species level with high accuracy and sensitivity (27,28). However, a favorable-quality DNA sample is initially required to amplify the full-length gene for long-read sequencing; therefore, one limitation of this approach is the difficulty of achieving full-length gene amplification in low-quality DNA samples. ...
End-stage kidney disease (ESKD) is the final stage of chronic kidney disease (CKD), in which long-term damage has been caused to the kidneys to the extent that they are no longer able to filter the blood of waste and extra fluid. Peritoneal dialysis (PD) is one of the treatments that remove waste products from the blood through the peritoneum which can improve the quality of life for patients with ESKD. However, PD-associated peritonitis is an important complication that contributes to the mortality of patients, and the detection of bacterial pathogens is associated with a high culture-negative rate. The present study aimed to apply a metagenomic approach for the bacterial identification in the PD effluent (PDE) of patients with CKD based on 16S ribosomal DNA sequencing. As a result of this investigation, five major bacteria species, namely Escherichia coli, Phyllobacterium myrsinacearum, Streptococcus gallolyticus, Staphylococcus epidermidis and Shewanella algae, were observed in PDE samples. Taken together, the findings of the present study have suggested that this metagenomic approach could provide a greater potential for bacterial taxonomic identification compared with traditional culture methods, suggesting that this is a practical and culture-independent alternative approach that will offer a novel preventative infectious strategy in patients with CDK.
... For microbiome profiling, 16S rRNA, which is the gold standard in microbial typing for bacteria and archaea, and 18S rRNA/ITS gene for fungi are first amplified by PCR with universal primers (Table 1) annealed to conserved regions and then sequenced. The sequencing data are subjected to bioinformatics analysis using freely available analysis tools for taxonomic classification (Quantitative Insights into Microbial Ecology (QIIME), Mothur, DADA2, Phyloseq, and METAGENassist) (Shahi et al. 2019) with the following three important steps: data pre-processing and quality management, taxonomic profiling, and community characterization, in which the variable regions are used to discriminate between bacterial taxa (Matsuo et al. 2021). ...
The human body harbors an extremely complex and dynamic microbial community (10-100 trillion) of bacteria, archaea, viruses, and eukaryotes. The human microbiota plays a crucial role in the environment and human health under normal circumstances; nonetheless, dysfunction of the human microbiome has been associated with illnesses ranging from inflammatory bowel disease to multidrug-resistant infections. Culture-dependent human microbial exploration approaches are inadequate for discovering the diversity, abundance, and full genetic and metabolic potential of the whole microbial ecosystem. Hence, metagenomics analysis is a powerful advanced technology for comprehensively studying human microbial composition and diversity, exploring novel and antibiotic resistance genes, microbial metabolic pathways, functional dysbiosis, and co-evolution of the microbiome with the host. Therefore, owing to the increase in human microbiome sequencing projects in healthy and diseased people worldwide, it is feasible to explore the human microbiome using a metagenomics approach. Thus, this review focuses on the advancement of metagenomics for exploring the human microbiome, human microbiome metagenomics data processing, and analysis strategies. Currently used bioinformatics tools and their approaches are also discussed in the context of human microbial metagenomics.
... However, third-generation sequencing platforms which can provide full-length 16S amplification in microbiome studies have seen rise in recent years, providing several advantages over short-read sequencing, including higher resolution in terms of diversity and taxonomic classification and the ability to detect additional taxa that may be missed by short-read sequencing [79]. Full-length 16S sequencing has been shown to provide species-level resolution in human gut microbiota studies [80]. However, full-length 16S sequencing also has limitations, including a higher cost and longer analysis time, and still may not be able to discriminate some closely related species [81]. ...
Pancreatic cancer (PC) ranks as the seventh leading cause of cancer-related deaths, with approximately 500,000 new cases reported in 2020. Existing strategies for early PC detection primarily target individuals at high risk of developing the disease. Nevertheless, there is a pressing need to identify innovative clinical approaches and personalized treatments for effective PC management. This study aimed to explore the dysbiosis signature of the fecal microbiota in PC and potential distinctions between its Intraductal papillary mucinous neoplasm (IPMN) and pancreatic ductal adenocarcinoma (PDAC) phenotypes, which could carry diagnostic significance. The study enrolled 33 participants, including 22 diagnosed with PDAC, 11 with IPMN, and 24 healthy controls. Fecal samples were collected and subjected to microbial diversity analysis across various taxonomic levels. The findings revealed elevated abundances of Firmicutes and Proteobacteria in PC patients, whereas healthy controls exhibited higher proportions of Bacteroidota. Both LEfSe and Random Forest analyses indicated the microbiome’s potential to effectively distinguish between PC and healthy control samples but fell short of differentiating between IPMN and PDAC samples. These results contribute to the current understanding of this challenging cancer type and highlight the applications of microbiome research. In essence, the study provides clear evidence of the gut microbiome’s capability to serve as a biomarker for PC detection, emphasizing the steps required for further differentiation among its diverse phenotypes.
... We agree that it is an excellent tool for rapid response monitoring of targeted organisms, particularly because of the read length. The kilobase and longer reads allow sequencing the full 16 S rRNA gene to achieve species-level resolution for bacteria [40]. Longer reads allow the variable fungal ITS marker to be flanked by more the more conservative 18 S and 28 S, which could aide phylogenetic placement and species level distinction for some taxa [37]. ...
Background
Soil microbial communities are difficult to measure and critical to soil processes. The bulk soil microbiome is highly diverse and spatially heterogeneous, which can make it difficult to detect and monitor the responses of microbial communities to differences or changes in management, such as different crop rotations in agricultural research. Sampling a subset of actively growing microbes should promote monitoring how soil microbial communities respond to management by reducing the variation contributed by high microbial spatial and temporal heterogeneity and less active microbes. We tested an in-growth bag method using sterilized soil in root-excluding mesh, “sterile sentinels,” for the capacity to differentiate between crop rotations. We assessed the utility of different incubation times and compared colonized sentinels to concurrently sampled bulk soils for the statistical power to differentiate microbial community composition in low and high diversity crop rotations. We paired this method with Oxford Nanopore MinION sequencing to assess sterile sentinels as a standardized, fast turn-around monitoring method.
Results
Compared to bulk soil, sentinels provided greater statistical power to distinguish between crop rotations for bacterial communities and equivalent power for fungal communities. The incubation time did not affect the statistical power to detect treatment differences in community composition, although longer incubation time increased total biomass. Bulk and sentinel soil samples contained shared and unique microbial taxa that were differentially abundant between crop rotations.
Conclusions
Overall, compared to bulk soils, the sentinels captured taxa with copiotrophic or ruderal traits, and plant-associated taxa. The sentinels show promise as a sensitive, scalable method to monitor soil microbial communities and provide information complementary to traditional soil sampling.
... Importantly, its application in fulllength 16S rRNA gene sequencing enables in-depth taxonomic classification (Oehler et al. 2023). Numerous studies have compared ONT LRS to Illumina based 16S rRNA gene sequencing in mock communities, swabs, and faecal samples (Shin et al. 2016;Acharya et al. 2019;Winand et al. 2019;Fujiyoshi et al. 2020;Heikema et al. 2020;Wei et al. 2020;de Siqueira et al. 2021;Low et al. 2021;Matsuo et al. 2021;Oberle et al. 2021;Park et al. 2021;Rozas et al. 2021;Szoboszlay et al. 2023;Connell et al. 2024). Four studies investigated the difference in beta-diversity (Heikema et al. 2020;de Siqueira et al. 2021;Szoboszlay et al. 2023;Connell et al. 2024), with two studies showing differences in beta-diversities between ONT and Illumina (Heikema et al. 2020;Szoboszlay et al. 2023). ...
... Two other studies measured sum of agreement at genera level (sum of the percentage of matching genera) and showed that the median microbiome agreement between ONT and Illumina groups ranged from 65 to 70% (Heikema et al. 2020;Connell et al. 2024). Moreover, many studies have analysed the differences or correlations between the abundance estimates generated by ONT and Illumina sequencing technologies at different taxonomic levels (Shin et al. 2016;Acharya et al. 2019;Winand et al. 2019;Fujiyoshi et al. 2020;Heikema et al. 2020;Wei et al. 2020;de Siqueira et al. 2021;Low et al. 2021;Matsuo et al. 2021;Oberle et al. 2021;Park et al. 2021;Rozas et al. 2021;Szoboszlay et al. 2023;Connell et al. 2024). The consensus is that at higher taxonomic levels, there were greater correlation observed, while the least correlation was observed at species level (Shin et al. 2016;Wei et al. 2020;Matsuo et al. 2021;Connell et al. 2024). ...
... Moreover, many studies have analysed the differences or correlations between the abundance estimates generated by ONT and Illumina sequencing technologies at different taxonomic levels (Shin et al. 2016;Acharya et al. 2019;Winand et al. 2019;Fujiyoshi et al. 2020;Heikema et al. 2020;Wei et al. 2020;de Siqueira et al. 2021;Low et al. 2021;Matsuo et al. 2021;Oberle et al. 2021;Park et al. 2021;Rozas et al. 2021;Szoboszlay et al. 2023;Connell et al. 2024). The consensus is that at higher taxonomic levels, there were greater correlation observed, while the least correlation was observed at species level (Shin et al. 2016;Wei et al. 2020;Matsuo et al. 2021;Connell et al. 2024). To date, there has been no comparison between LRS and SRS using tumour tissue samples. ...
Describing the microbial community within the tumour has been a key aspect in understanding the pathophysiology of the tumour microenvironment. In head and neck cancer (HNC), most studies on tissue samples have only performed 16S rRNA short-read sequencing (SRS) on V3-V5 region. SRS is mostly limited to genus level identification. In this study, we compared full-length 16S rRNA long-read sequencing (FL-ONT) from Oxford Nanopore Technology (ONT) to V3-V4 Illumina SRS (V3V4-Illumina) in 26 HNC tumour tissues. Further validation was also performed using culture-based methods in 16 bacterial isolates obtained from 4 patients using MALDI-TOF MS. We observed similar alpha diversity indexes between FL-ONT and V3V4-Illumina. However, beta-diversity was significantly different between techniques (PERMANOVA - R² = 0.131, p < 0.0001). At higher taxonomic levels (Phylum to Family), all metrics were more similar among sequencing techniques, while lower taxonomy displayed more discrepancies. At higher taxonomic levels, correlation in relative abundance from FL-ONT and V3V4-Illumina were higher, while this correlation decreased at lower levels. Finally, FL-ONT was able to identify more isolates at the species level that were identified using MALDI-TOF MS (75% vs. 18.8%). FL-ONT was able to identify lower taxonomic levels at a better resolution as compared to V3V4-Illumina 16S rRNA sequencing.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00203-024-03985-7.
