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Outbreaks of furunculosis cause significant losses in salmonid aquaculture worldwide. With a recent rise in antimicrobial resistance, regulatory measures to minimize the use of antibiotics in animal husbandry, including aquaculture, have increased scrutiny and availability of veterinary medical products to control this disease in production facilities. In such a regulatory environment, the utility of autogenous vaccines to assist with disease prevention and control as a veterinary-guided prophylactic measure is of high interest to the producers and veterinary services alike. However, evolving concepts of epidemiological units and epidemiological links need to be considered during approval and acceptance procedures for the application of autogenous vaccines in multiple aquaculture facilities. Here, we present the results of solid-state nanopore sequencing (Oxford Nanopore Technologies, ONT) performed on 54 isolates of Aeromonas salmonicida ssp. salmonicida sampled during clinical outbreaks of furunculosis in different aquaculture facilities from Bavaria, Germany, from 2017 to 2020. All of the performed analyses (phylogeny, single nucleotide polymorphism and 3D protein modeling for major immunogenic proteins) support a high probability that all studied isolates belong to the same epidemiological unit. Simultaneously, we describe a cost/effective method of whole genome analysis with the usage of ONT as a viable strategy to study outbreaks of other pathogens in the field of aquatic veterinary medicine for the purpose of developing the best autogenous vaccine candidates applicable to multiple aquaculture establishments. [ABSTRACT FROM AUTHOR]

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined. [ABSTRACT FROM AUTHOR]

Background. In the context of increasing antimicrobial resistance (AMR), whole-genome sequencing (WGS) of bacteria is considered a highly accurate and comprehensive surveillance method for detecting and tracking the spread of resistant pathogens. Two primary sequencing technologies exist: short-read sequencing (50–300 base pairs) and long-read sequencing (thousands of base pairs). The former, based on Illumina sequencing platforms (ISPs), provides extensive coverage and high accuracy for detecting single nucleotide polymorphisms (SNPs) and small insertions/deletions, but is limited by its read length. The latter, based on platforms such as Oxford Nanopore Technologies (ONT), enables the assembly of genomes, particularly those with repetitive regions and structural variants, although its accuracy has historically been lower. Results. We performed a head-to-head comparison of these techniques to sequence the K. pneumoniae VS17 isolate, focusing on blaNDM resistance gene alleles in the context of a surveillance program. Discrepancies between the ISP (blaNDM-4 allele identified) and ONT (blaNDM-1 and blaNDM-5 alleles identified) were observed. Conjugation assays and Sanger sequencing, used as the gold standard, confirmed the validity of ONT results. This study demonstrates the importance of long-read or hybrid assemblies for accurate carbapenemase resistance gene identification and highlights the limitations of short reads in the context of gene duplications or multiple alleles. Conclusions. In this proof-of-concept study, we conclude that recent long-read sequencing technology may outperform standard short-read sequencing for the accurate identification of carbapenemase alleles. Such information is crucial given the rising prevalence of strains producing multiple carbapenemases, especially as WGS is increasingly used for epidemiological surveillance and infection control. [ABSTRACT FROM AUTHOR]

Brassica oleracea has been developed into many important crops, including cabbage, kale, cauliflower, broccoli and so on. The genome and gene annotation of cabbage (cultivar JZS), a representative morphotype of B. oleracea, has been widely used as a common reference in biological research. Although its genome assembly has been updated twice, the current gene annotation still lacks information on untranslated regions (UTRs) and alternative splicing (AS). Here, we constructed a high-quality gene annotation (JZSv3) using a full-length transcriptome acquired by nanopore sequencing, yielding a total of 59 452 genes and 75 684 transcripts. Additionally, we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference, and found that 3843 out of 11 908 differentially expressed genes (DEGs) underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress. Meanwhile, we also identified many AS genes, including BolLHCB5 and BolHSP70, that displayed distinct expression patterns within variant transcripts of the same gene, highlighting the importance of JZSv3 as a pivotal reference for AS analysis. Overall, JZSv3 provides a valuable resource for exploring gene function, especially for obtaining a deeper understanding of AS regulation mechanisms.

To reduce the use of antibiotics and chemicals in aquaculture, an edible herb, Bidens pilosa, has been selected as a multifunctional feed additive. Although there has been considerable research into the effects of B. pilosa on poultry, the wider effects of B. pilosa, particularly on the growth and gut microbiota of fish, remain largely unexplored. We aimed to investigate the interactive effects between the host on growth and the gut microbiota using transcriptomics and the gut microbiota in B. pilosa-fed tilapia. In this study, we added 0.5% and 1% B. pilosa to the diet and observed that the growth performance of tilapia significantly increased over 8 weeks of feeding. Comparative transcriptome analysis was performed on RNA sequence profiles obtained from liver and muscle tissues. Functional enrichment analysis revealed that B. pilosa regulates several pathways and genes involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, endocrine system, signal transduction, and metabolism of other amino acids. The expression of the selected growth-associated genes was validated by qRT–PCR. The qRT–PCR results indicated that B. pilosa may enhance growth performance by activating the expression of the liver igf1 and muscle igf1rb genes and inhibiting the expression of the muscle negative regulator mstnb. Both the enhancement of liver endocrine IGF1/IGF1Rb signaling and the suppression of muscle autocrine/paracrine MSTN signaling induced the expression of myogenic regulatory factors (MRFs), myod1, myog and mrf4 in muscle to promote muscle growth in tilapia. The predicted function of the gut microbiota showed several significantly different pathways that overlapped with the KEGG enrichment results of differentially expressed genes in the liver transcriptomes. This finding suggested that the gut microbiota may influence liver metabolism through the gut–liver axis in B. pilosa-fed tilapia. In conclusion, dietary B. pilosa can regulate endocrine IGF1 signaling and autocrine/paracrine MSTN signaling to activate the expression of MRFs to promote muscle growth and alter the composition of gut bacteria, which can then affect liver amino acid metabolism, carbohydrate metabolism, endocrine system, lipid metabolism, metabolism of other amino acids, and signal transduction in the host, ultimately enhancing growth performance. Our results suggest that B. pilosa has the potential to be a functional additive that can be used as an alternative to reduce antibiotic use as a growth promoter in aquaculture. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Pathology is the property of Canadian Association of Pathologists and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent–child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types. [ABSTRACT FROM AUTHOR]

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies. • ONT Q20 + and dual UMIs enable accurate long-read sequencing in antibody selection. • ONT-UMI monitors diversity/enrichment throughout antibody phage-display rounds. • Compared to colony-picking, ONT-UMI recovers rare binders even at 0,3% abundant. • ONT-UMI reveals binding motifs for affinity maturation and specificity tuning. [ABSTRACT FROM AUTHOR]

The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages responsible for their widespread diffusion, these methods have some constraints, mainly related to short read length and the need for PCR amplification. As a consequence, long-read sequencers, called third-generation sequencing (TGS), have been developed, promising to overcome NGS. Starting from the first prototype, TGS has progressively ameliorated its chemistries by improving both read length and base-calling accuracy, as well as simultaneously reducing the costs/base. Based on these premises, TGS is showing its potential in many fields, including the analysis of difficult-to-sequence genomic regions, structural variations detection, RNA expression profiling, DNA methylation study, and metagenomic analyses. Protocol standardization and the development of easy-to-use pipelines for data analysis will enhance TGS use, also opening the way for their routine applications in diagnostic contexts. [ABSTRACT FROM AUTHOR]

الملخص: أهدف البحث: تعكس عدد حالات كورونا في إندونيسيا خطورة الأمراض وانتشارها بسرعة. تم إجراء الرصد الجينومي لفيروس كورونا المستجد والتحقيق في المجتمعات البكتيرية للجهاز التنفسي العلوي في غرب جاوة كرد فعل للتهديد المتزايد حيث يمكن أن يؤثر تفاوت التوازن في الميكروبات في الجهاز التنفسي العلوي بشكل سلبي على حالة المرضى. طرق البحث: استخدمنا منصة تسلسل النانوبور لتحليل 43 عينة من التغيرات الجينية لفيروس كورونا المستجد و11 عينة مختارة لتسلسل الجينات عبر الريبوزوم 16، تم جمعهم في الفترة من مايو إلى أغسطس 2021. النتائج: خمسة أنواع فيروسية في السكان (أ.ي.23؛ أ.ي.24؛ أ.ي.26؛ أ.ي.42؛ ب.1.1.7) كانت تهيمن على وجود النوع أ.ي.23 (82%) predominated among five virus lineages in the populations (AY.23, AY.24, AY.26, AY.42, B.1.1.7). The region in the SARS-CoV-2 genome found to have the highest number of mutations was the spike (S) protein (>20%). There was no association between SARS-CoV-2 lineages, mutation frequency, patient profile, and COVID-19 rapid spread-categorized cases. There was no association of bacterial relative abundance, alpha-beta diversity, and linear discriminant analysis effect size analysis with patient profile and rapid spread cases. MetagenomeSeq analysis showed eight differential abundance species in individual patient profiles, including Pseudomonas aeruginosa and Haemophilus parainfluenzae. Conclusions: The data demonstrated relevant AY.23 dominance (the Delta variant) in West Java during that period supporting the importance of surveillance program in monitoring disease progression. The inconsistent results of the bacterial communities suggest that a complex multifactor process may contribute to the progression of bacterial-induced disease in each patient.

During the recent avian influenza epizootics that occurred in France in 2020/21 and 2021/22, the virus was so contagiousness that it was impossible to control its spread between farms. The preventive slaughter of millions of birds consequently was the only solution available. In an effort to better understand the spread of avian influenza viruses (AIVs) in a rapid and innovative manner, we established an amplicon-based MinION sequencing workflow for the rapid genetic typing of circulating AIV strains. An amplicon-based MinION sequencing workflow based on a set of PCR primers targeting primarily the hemagglutinin gene but also the entire influenza virus genome was developed. Thirty field samples from H5 HPAIV outbreaks in France, including environmental samples, were sequenced using the MinION MK1C. A real-time alignment of the sequences with MinKNOW software allowed the sequencing run to be stopped as soon as enough data were generated. The consensus sequences were then generated and a phylogenetic analysis was conducted to establish links between the outbreaks. The whole sequence of the hemagglutinin gene was obtained for the 30 clinical samples of H5Nx HPAIV belonging to clade 2.3.4.4b. The consensus sequences comparison and the phylogenetic analysis demonstrated links between some outbreaks. While several studies have shown the advantages of MinION for avian influenza virus sequencing, this workflow has been applied exclusively to clinical field samples, without any amplification step on cell cultures or embryonated eggs. As this type of testing pipeline requires only a short amount of time to link outbreaks or demonstrate a new introduction, it could be applied to the real-time management of viral epizootics. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Taibah University Medical Sciences is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this comprehensive review, we have explored the role of Oxford Nanopore Technologies (ONT) in the sequencing of the SARS-CoV-2 genome, highlighting its significant contributions, methodologies, achievements, and challenges. ONT stands out for its real-time sequencing capabilities, portability, and long-read sequencing, providing crucial insights into the virus's genetic makeup, aiding in the rapid identification of variants, and enhancing global epidemiological surveillance. Despite challenges such as higher error rates and complex data analysis requirements, ongoing advancements in nanopore technology and computational methods are steadily overcoming these hurdles. Looking forward, the integration of artificial intelligence and machine learning, along with the expansion of ONT's applications into areas like clinical diagnostics and personalized medicine, are poised to further revolutionize genomic research. This review underscores the importance of ONT in current and future genomic studies, particularly in understanding and combating infectious diseases like COVID-19, and highlights its potential in addressing global health challenges. [ABSTRACT FROM AUTHOR]

Background. In the context of increasing antimicrobial resistance (AMR), whole-genome sequencing (WGS) of bacteria is considered a highly accurate and comprehensive surveillance method for detecting and tracking the spread of resistant pathogens. Two primary sequencing technologies exist: short-read sequencing (50–300 base pairs) and long-read sequencing (thousands of base pairs). The former, based on Illumina sequencing platforms (ISPs), provides extensive coverage and high accuracy for detecting single nucleotide polymorphisms (SNPs) and small insertions/deletions, but is limited by its read length. The latter, based on platforms such as Oxford Nanopore Technologies (ONT), enables the assembly of genomes, particularly those with repetitive regions and structural variants, although its accuracy has historically been lower. Results. We performed a head-to-head comparison of these techniques to sequence the K. pneumoniae VS17 isolate, focusing on blaNDM resistance gene alleles in the context of a surveillance program. Discrepancies between the ISP (blaNDM-4 allele identified) and ONT (blaNDM-1 and blaNDM-5 alleles identified) were observed. Conjugation assays and Sanger sequencing, used as the gold standard, confirmed the validity of ONT results. This study demonstrates the importance of long-read or hybrid assemblies for accurate carbapenemase resistance gene identification and highlights the limitations of short reads in the context of gene duplications or multiple alleles. Conclusions. In this proof-of-concept study, we conclude that recent long-read sequencing technology may outperform standard short-read sequencing for the accurate identification of carbapenemase alleles. Such information is crucial given the rising prevalence of strains producing multiple carbapenemases, especially as WGS is increasingly used for epidemiological surveillance and infection control.

During the recent avian influenza epizootics that occurred in France in 2020/21 and 2021/22, the virus was so contagiousness that it was impossible to control its spread between farms. The preventive slaughter of millions of birds consequently was the only solution available. In an effort to better understand the spread of avian influenza viruses (AIVs) in a rapid and innovative manner, we established an amplicon-based MinION sequencing workflow for the rapid genetic typing of circulating AIV strains. An amplicon-based MinION sequencing workflow based on a set of PCR primers targeting primarily the hemagglutinin gene but also the entire influenza virus genome was developed. Thirty field samples from H5 HPAIV outbreaks in France, including environmental samples, were sequenced using the MinION MK1C. A real-time alignment of the sequences with MinKNOW software allowed the sequencing run to be stopped as soon as enough data were generated. The consensus sequences were then generated and a phylogenetic analysis was conducted to establish links between the outbreaks. The whole sequence of the hemagglutinin gene was obtained for the 30 clinical samples of H5Nx HPAIV belonging to clade 2.3.4.4b. The consensus sequences comparison and the phylogenetic analysis demonstrated links between some outbreaks. While several studies have shown the advantages of MinION for avian influenza virus sequencing, this workflow has been applied exclusively to clinical field samples, without any amplification step on cell cultures or embryonated eggs. As this type of testing pipeline requires only a short amount of time to link outbreaks or demonstrate a new introduction, it could be applied to the real-time management of viral epizootics.

The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages responsible for their widespread diffusion, these methods have some constraints, mainly related to short read length and the need for PCR amplification. As a consequence, long-read sequencers, called third-generation sequencing (TGS), have been developed, promising to overcome NGS. Starting from the first prototype, TGS has progressively ameliorated its chemistries by improving both read length and base-calling accuracy, as well as simultaneously reducing the costs/base. Based on these premises, TGS is showing its potential in many fields, including the analysis of difficult-to-sequence genomic regions, structural variations detection, RNA expression profiling, DNA methylation study, and metagenomic analyses. Protocol standardization and the development of easy-to-use pipelines for data analysis will enhance TGS use, also opening the way for their routine applications in diagnostic contexts.

Austropuccinia psidii is the causal agent of myrtle rust in over 480 species within the family Myrtaceae. Lineages of A. psidii are structured by their hosts in the native range, and some have success in infecting newly encountered hosts. For example, the pandemic biotype has spread beyond South America, and proliferation of other lineages is an additional risk to biodiversity and industries. Efforts to manage A. psidii incursions, including lineage differentiation, relies on variable microsatellite markers. Testing these markers is time-consuming, complex, and requires reference material that is not always readily available. We designed a novel diagnostic approach targeting eight selectively chosen loci including the fungal mating-type HD (homeodomain) transcription factor locus. The HD locus ( bW1/2-HD1 and bE1/2-HD2 ) is highly polymorphic, facilitating clear biological predictions about its inheritance from founding populations. To be considered as potentially derived from the same lineage, all four HD alleles must be identical. If all four HD alleles are identical six additional markers can further differentiate lineage identity. Our lineage diagnostics relies on PCR amplification of eight loci in different genotypes of A. psidii followed by amplicon sequencing using Oxford Nanopore Technologies (ONT) and comparative analysis. The lineage-specific assay was validated on four isolates with existing genomes, uncharacterized isolates, and directly from infected leaf material. We reconstructed alleles from amplicons and confirmed their sequence identity relative to their reference. Genealogies of alleles confirmed the variations at the loci among lineages/isolates. Our study establishes a robust diagnostic tool for differentiating known lineages of A. psidii based on biological predictions and available nucleotide sequences. This tool is suited to detecting the origin of new pathogen incursions.

To comprehend the effects of potentially invasive coral Tubastraea aurea on marine ecosystems, it is crucial to understand their adaptive strategies to survive environmental changes and perturbations. Therefore, a cross-transplantation study was conducted to assess the microbiome's role in the resilience of T. aurea to sudden environmental changes.Hydrographic analyses revealed distinct ecological conditions at two sites: a hydrothermal vent (HV) site, characterized by harsh environmental conditions serving as a natural laboratory for future oceanic changes, and a regular coastal site Fulong (FU). Both sites showed significant differences in pH, temperature, and dissolved oxygen. Using Oxford Nanopore Technologies, we examined bacterial dynamics in coral tissue, mucus and ambient sediment samples following cross-transplantation experiments. We observed a rapid shift in dominant bacterial groups post-transplantation with transplanted corals acquiring microbiomes similar to native corals from their respective sites within 16 days. The bacteria Endozoicomonas euniceicola and Ruegeria profundi were dominant in both native and transplanted corals, suggesting their critical role in coral resilience. Furthermore, the enrichment of certain bacterial taxa post-transplantation suggests that opportunistic species also contribute to host acclimatization. Functional profiling data indicated that there was site-specific adaptation because corals had acquired beneficial bacterial assemblages to assist them cope with environmental stressors. More specifically, there was a switch towards sulfur and nitrogen metabolism in corals that moved to high sulfidic environments, while corals transplanted into normal coastal environments showed enriched photoautotrophic processes due to their symbionts. Our study underscored the highly flexible microbiome of T. aurea and its pivotal role in facilitating host resilience to environmental perturbations, particularly in the context of its potential invasiveness. Hence, these findings contribute to the understanding of coral-microbiome dynamics and emphasize the necessity of considering microbially-mediated resilience in managing potentially invasive coral species in marine ecosystems around the world, especially as ocean conditions continue to change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Vibrio sp. NTOU- M3 is a potential new bacterium isolated from hard clam ( Meretrix taiwanica ) in the estuarine region of Taiwan. The complete sequences obtained using Oxford Nanopore Technologies and Illumina sequencing consist of a 3,272,438-bp large circular chromosome and a 1,584,497-bp small circular chromosome.

The data presented in this study are metagenomic profiles of human gut microbiome deduced from frozen fecal samples using two different sequencing chemistries namely, Illumina and Oxford Nanopore Technologies (ONT). The generated data is obtained from genomic DNA extracted from frozen fecal samples collected from a healthy individual on Day 3, Day 5, Day 9, Day 12, and Day 30, in addition to Day 1 (unfrozen). The metagenomic sequence data have been deposited at NCBI SRA as BioProject PRJNA827663. The taxonomic annotation undertaken using MG-RAST showed relative abundance of bacteria represented by different taxonomic levels varied significantly based on two sequencing chemistries. There was distinct temporal variation in the relative abundance of bacteria at different taxonomic levels based on the day of extraction of genomic DNA. This dataset can be used to study differences in functional profiles of human gut microbiome using different sequencing technologies. Moreover, generated data can aid in selection of most appropriate sequencing chemistry to study future human gut microbiome studies based on the appropriate preservation method of fecal samples., (© 2024 Published by Elsevier Inc.)

Rapid advancements in sequencing technologies have led to significant progress in microbial genomics, yet challenges persist in accurately identifying microbial strain diversity in metagenomic samples, especially when working with noisy long-read data from platforms like Oxford Nanopore Technologies (ONT). In this article, we introduce NanoMGT, a tool designed to enhance marker gene typing in low-complexity mono-species samples, leveraging the unique properties of long reads. NanoMGT excels in its ability to accurately identify mutations amidst high error rates, ensuring the reliable detection of multiple strain-specific marker genes. Our tool implements a novel scoring system that rewards mutations co-occurring across different reads and penalizes densely grouped, likely erroneous variants, thereby achieving a good balance between sensitivity and precision. A comparative evaluation of NanoMGT, using a simulated multi-strain sample of seven bacterial species, demonstrated superior performance relative to existing tools and the advantages of using a threshold-based filtering approach to calling minority variants in ONT's sequencing data. NanoMGT's potential as a post-binning tool in metagenomic pipelines is particularly notable, enabling researchers to more accurately determine specific alleles and understand strain diversity in microbial communities. Our findings have significant implications for clinical diagnostics, environmental microbiology, and the broader field of genomics. The findings offer a reliable and efficient approach to marker gene typing in complex metagenomic samples., (© The Author(s) 2024. Published by Oxford University Press.)

16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species-level resolution. This pipeline provides a low-cost solution for characterizing microbiome composition by exploiting real-time, long-read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full-length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu., (© 2024 Wiley Periodicals LLC.)