Search

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]

Gene therapies using recombinant adeno-associated virus (AAV) vectors have demonstrated considerable clinical success in the treatment of genetic disorders. Improved vectors with favorable tropism profiles, decreased immunogenicity, and enhanced manufacturability are poised to further improve the state of gene therapies. Such vectors can be identified through directed evolution, a process of subjecting a diverse capsid library to a selection pressure to identify individual variants with a desired trait. Currently, libraries that involve changes distributed throughout the AAV capsid coding region, such as DNA family shuffled libraries, are largely characterized using low-throughput Sanger sequencing of individual clones. However, improvements in long-read sequencing technologies have increased their applicability to capsid libraries and evaluation of the selection process. Here, we explore the application of Oxford Nanopore Technologies refined by a concatemeric consensus method for initial library characterization and monitoring selection of a shuffled AAV capsid library. Furthermore, we present AAVolve, a bioinformatic pipeline for processing long-read data from AAV-directed evolution experiments. Our approach allows high-throughput characterization of AAV capsids in a streamlined manner, facilitating deeper insights into library composition through multiple rounds of selection, and generalization through training of machine learning models.

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]

Several species of sacoglossan sea slugs possess the incredible ability to sequester chloroplasts from the algae they consume. These "photosynthetic animals" incorporate stolen chloroplasts, called kleptoplasts, into the epithelial cells of tubules that extend from their digestive tracts throughout their bodies. The mechanism by which these slugs maintain functioning kleptoplasts in the absence of an algal nuclear genome is unknown. Here, we report a draft genome of the sacoglossan slug Elysia crispata morphotype clarki, a morphotype native to the Florida Keys that can retain photosynthetically active kleptoplasts for several months without feeding. We used a combination of Oxford Nanopore Technologies long reads and Illumina short reads to produce a 786-Mb assembly (N50 = 0.459 Mb) containing 68,514 predicted protein-coding genes. A phylogenetic analysis found no evidence of horizontal acquisition of genes from algae. We performed gene family and gene expression analyses to identify E. crispata genes unique to kleptoplast-containing slugs that were more highly expressed in fed versus unfed developmental life stages. Consistent with analyses in other kleptoplastic slugs, our investigation suggests that genes encoding lectin carbohydrate-binding proteins and those involved in regulation of reactive oxygen species and immunity may play a role in kleptoplast retention. Lastly, we identified four polyketide synthase genes that could potentially encode proteins producing UV- and oxidation-blocking compounds in slug cell membranes. The genome of E. crispata is a quality resource that provides potential targets for functional analyses and enables further investigation into the evolution and mechanisms of kleptoplasty in animals. [ABSTRACT FROM AUTHOR]

Abstract Background Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. Results Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. Conclusions Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

Abstract Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.

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 Background Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications. Methods We used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth. Results Across 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis. Conclusions We present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey.

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.

With the increasing occurrence and severity of cyanobacterial harmful algal blooms (cHAB) at the global scale, there is an urgent need for rapid, accurate, accessible, and cost-effective detection tools. Here, we detail the RosHAB workflow, an innovative, in-the-field applicable genomics approach for real-time, early detection of cHAB outbreaks. We present how the proposed workflow offers consistent taxonomic identification of water samples in comparison to traditional microscopic analyses in a few hours and discuss how the generated data can be used to deepen our understanding on cyanobacteria ecology and forecast HABs events. In parallel, processed water samples will be used to iteratively build the International cyanobacterial toxin database (ICYATOX; http://icyatox.ibis.ulaval. ca) containing the analysis of novel cyanobacterial genomes, including phenomics and genomics metadata. Ultimately, RosHAB will (1) improve the accuracy of on-site rapid diagnostics, (2) standardize genomic procedures in the field, (3) facilitate these genomics procedures for non-scientific personnel, and (4) identify prognostic markers for evidence-based decisions in HABs surveillance. [ABSTRACT FROM AUTHOR]

Background: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. Results: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. Conclusions: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens. [ABSTRACT FROM AUTHOR]

Background: This study aimed to perform preimplantation genetic testing (PGT) for a female Coffin-Lowry Syndrome (CLS) patient with a de novo mutation (DNM) in RPS6KA3. It was challenging to establish the haplotype in this family because of the lack of information from affected family members. Hence, we explored a new and reliable strategy for the detection of the DNM in PGT, using Oxford Nanopore Technologies (ONT) and the MARSALA platform. Methods: We performed whole-exome sequencing (WES) on the proband and confirmed the pathogenic mutation by Sanger sequencing. The proband then underwent PGT to prevent the transmission of the pathogenic mutation to her offspring. We diverged from the conventional methods and used long-read sequencing (LRS) on the ONT platform to directly detect the mutation and nearby SNPs, for construction of the haplotype in the preclinical phase of PGT. In the clinical phase of embryo diagnosis, the MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations (CNVs), in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus. Sanger sequencing and karyotyping were performed by amniocentesis, at 17 weeks of gestation, to confirm the accuracy of PGT. Results: Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of RPS6KA3 in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby.Conclusion: The ONT platform, combined with the MARSALA method, can be used to perform PGT for DNM patients without the need for other samples as a reference. [ABSTRACT FROM AUTHOR]

Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys. [ABSTRACT FROM AUTHOR]

Emerging evidence has recently revealed a prominent role of the microbiome in pancreatic ductal adenocarcinoma (PDAC). However, while most observations were made in patients, mouse models still require a precise characterization of their disease-related microbiome to employ them for mechanistic and interventional preclinical studies. To investigate the fecal and tumoral microbiome of LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) and control (CTRL) mice, Oxford Nanopore sequencing was applied. Feces were collected from 10 KPC mice and 10 CTRLs at 3 timepoints (6 weeks, 12 weeks, and when tumor-bearing (KPC) or 6 months (CTRL), respectively). Metagenomic sequencing was performed on feces DNA. KPC tumor and healthy pancreas DNA samples were subjected to 16S rRNA gene sequencing. Bacterial marker components were detected in KPC tumor tissue over time by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Murine fecal samples showed a significantly different microbiome compared to age-matched healthy CTRLs regarding beta diversity (p = 0.001, R2 = 0.2–0.25 for Bray-Curtis). Adjusted human PDAC classifiers predicted disease status from feces of KPC mice achieving area under the receiver operating characteristic (AUROC) values of 80%. Furthermore, KPC tumors harbored significantly more bacterial components than healthy pancreas. Also the microbial composition differs significantly between KPC tumors and healthy pancreas tissue (p = 0.042 for Bray-Curtis). Microbiota found highly abundant in human PDAC samples were considerably more abundant in KPC tumors as compared to healthy pancreas samples (p-value <0.001). KPC fecal samples show similarities with the microbial composition of stool samples from human PDAC patients. [ABSTRACT FROM AUTHOR]

Xin Zhao,1,2,&ast; Yue Ge,1,3,&ast; Yuan Zhang,1 WenJie Zhang,1 HongBin Hu,1 LuLan Li,1 Tong Sha,1 ZhenHua Zeng,1 Feng Wu,1 ZhongQing Chen1 1Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2The First Clinical Medical School, Southern Medical University, Guangzhou, People’s Republic of China; 3Department of School of Nursing, Southern Medical University, Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: ZhongQing Chen; Feng Wu, Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China, Tel +86 13503049103 ; +86 15915751705, Email zhongqingchen2008@163.com; wishuhappy@126.comBackground: Next-generation sequencing of the metagenome (mNGS) is increasingly used in pathogen diagnosis for infectious diseases due to its short detection time. The time for Oxford Nanopore Technologies (ONT) sequencing-based etiology detection is further shortened compared with that of mNGS, but only a few studies have verified the time advantage and accuracy of ONT sequencing for etiology diagnosis. In 2022, a study confirmed that there was no significant difference in sensitivity and specificity between ONT and mNGS in suspected community-acquired pneumonia patients, which there was no clinical study verified in patients with SHAP.Methods: From October 24 to November 20, 2022, 10 patients with severe hospital-acquired pneumonia (SHAP) in the Nanfang Hospital intensive care unit (ICU) were prospectively enrolled. Bronchoalveolar lavage fluid (BALF) was collected for ONT sequencing, mNGS, and traditional culture. The differences in pathogen detection time and diagnostic agreement among ONT sequencing, mNGS, traditional culture method, and clinical composite diagnosis were compared.Results: Compared with mNGS and the traditional culture method, ONT sequencing had a significant advantage in pathogen detection time (9.6± 0.7 h versus 24.7± 2.7 h versus 132± 58 h, P < 0.05). The agreement rate between ONT sequencing and the clinical composite diagnosis was 73.3% (kappa value=0.737, P < 0.05).Conclusion: ONT sequencing has a potential advantage for rapidly identifying pathogens.Keywords: Oxford Nanopore Technologies, Nanopore sequencing, metagenomic next-generation sequencing, severe hospital-acquired pneumonia, pathogen

Abstract Background Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. Results Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. Conclusions Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

Abstract Background RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. Results We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Conclusion Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

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.

With the increasing occurrence and severity of cyanobacterial harmful algal blooms (cHAB) at the global scale, there is an urgent need for rapid, accurate, accessible, and cost-effective detection tools. Here, we detail the RosHAB workflow, an innovative, in-the-field applicable genomics approach for real-time, early detection of cHAB outbreaks. We present how the proposed workflow offers consistent taxonomic identification of water samples in comparison to traditional microscopic analyses in a few hours and discuss how the generated data can be used to deepen our understanding on cyanobacteria ecology and forecast HABs events. In parallel, processed water samples will be used to iteratively build the International cyanobacterial toxin database (ICYATOX; http://icyatox.ibis.ulaval.ca) containing the analysis of novel cyanobacterial genomes, including phenomics and genomics metadata. Ultimately, RosHAB will (1) improve the accuracy of on-site rapid diagnostics, (2) standardize genomic procedures in the field, (3) facilitate these genomics procedures for non-scientific personnel, and (4) identify prognostic markers for evidence-based decisions in HABs surveillance.

The Indo-Pacific native azooxanthellate Tubastraea (Scleractinia) has been identified as an invasive marine species with substantial environmental, economic, and social implications worldwide. Despite their exceptional invasive capacity, our understanding of the role of their symbiotic microbiota in host resilience, as well as their response to ambient environmental conditions, remains limited. In this study, we analyzed the symbiotic bacterial communities found in the tissue and mucus of Tubastraea aurea from different habitats along the northeastern coast of Taiwan. These habitats included two extreme sites (a hydrothermal vent [HV] and a copper mining [CM] site) and two normal environments (inlet of a nuclear power plant [NPP] and a habitat adjacent to a conservation zone [CZ]). We employed full-length 16S rRNA sequencing (~1.5 kilobases) to determine coral-associated microbiome responses to local environments. Results showed significant variations in bacterial communities between corals from extreme and normal habitats. Chemoheterotrophic Endozoicomonas bacteria dominated the tissue samples from the HV and CM sites, whereas phototrophic Synechococcus cyanobacteria dominated the NPP and CZ sites. Hydrographic parameters such as pH, salinity, biological oxygen demand, turbidity, and concentration of heavy metals (e.g., Cu and Fe) increased at the HV and CM sites compared with those at the NPP and CZ sites. This difference created more stressful conditions at the HV and CM sites. The microbial assemblages associated with T. aurea exhibited a prevalence of diverse symbiotic bacteria that could potentially contribute to the host’s ability to adapt and survive in challenging ecological conditions. Therefore, these advantageous microorganisms, along with the host’s physiological mechanisms of dispersion, range expansion, and invasiveness, may enhance the resilience and ability of T. aurea to thrive in extreme environments.

HLA allelic variation has been well studied and documented in many parts of the world. However, African populations have been relatively under‐represented in studies of HLA variation. We have characterized HLA variation from 489 individuals belonging to 13 ethnically diverse populations from rural communities from the African countries of Botswana, Cameroon, Ethiopia, and Tanzania, known to practice traditional subsistence lifestyles using next generation sequencing (Illumina) and long‐reads from Oxford Nanopore Technologies. We identified 342 distinct alleles among the 11 HLA targeted genes: HLA‐A, ‐B, ‐C, ‐DRB1, ‐DRB3, ‐DRB4, ‐DRB5, ‐DQA1, ‐DQB1, ‐DPA1, and ‐DPB1, with 140 of those alleles containing novel sequences that were submitted to the IPD‐IMGT/HLA database. Sixteen of the 140 alleles contained novel content within the exonic regions of the genes, while 110 alleles contained novel intronic variants. Four alleles were found to be recombinants of already described HLA alleles and 10 alleles extended the sequence content of already described alleles. All 140 alleles include complete allelic sequence from the 5′ UTR to the 3′ UTR that are inclusive of all exons and introns. This report characterizes the HLA allelic variation from these individuals and describes the novel allelic variation present within these specific African populations. [ABSTRACT FROM AUTHOR]

Thirty years after its designation as a federally endangered species, the Florida Perforate Cladonia (FPC) remains imperiled in isolated populations in the Florida scrub in the southeastern USA. For threatened and endangered species, such as FPC, reference genomes provide critical insight into genomic diversity, local adaptations, landscape-level genetics, and phylogenomics. Using high-throughput sequencing, we assemble the first draft nuclear and mitochondrial genomes for the FPC mycobiont—Cladonia perforata. We also assess genetic diversity within and among populations in southeastern Florida using genome-scale data and investigate diversity across the entire nuclear ribosomal cistron, including the standard DNA barcoding marker for fungi. The draft nuclear genome spanned 33.6 Mb, and the complete, circular mitochondrial genome was 59 Kb. We also generated the first chloroplast genome, to our knowledge, for the photobiont genus associated with FPC, an undescribed Asterochloris species. We inferred the presence of multiple, distinct mycobiont parental genotypes (genets) occurring at local scales in southeastern Florida, and strikingly, no genets were shared among even the closest sample sites. All sampled thalli shared identical mitochondrial genomes, while the nuclear ribosomal cistron showed limited variability—highlighting the genetic resolution provided by nuclear genome-scale datasets. The genomic resources generated here provide critical resources for informed conservation efforts for the FPC. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications. Methods: We used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth. Results: Across 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis. Conclusions: We present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey. [ABSTRACT FROM AUTHOR]

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Abstract Background Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT’s improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. Results Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy’s plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). Conclusions The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.

Background: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. Results: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. Conclusions: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future. [ABSTRACT FROM AUTHOR]

There is growing interest in the role of structural variants (SVs) as drivers of local adaptation and speciation. From a biodiversity genomics perspective, the characterization of genome‐wide SVs provides an exciting opportunity to complement single nucleotide polymorphisms (SNPs). However, little is known about the impacts of SV discovery and genotyping strategies on the characterization of genome‐wide SV diversity within and among populations. Here, we explore a near whole‐species resequence data set, and long‐read sequence data for a subset of highly represented individuals in the critically endangered kākāpō (Strigops habroptilus). We demonstrate that even when using a highly contiguous reference genome, different discovery and genotyping strategies can significantly impact the type, size and location of SVs characterized genome‐wide. Further, we found that the mean number of SVs in each of two kākāpō lineages differed both within and across generations. These combined results suggest that genome‐wide characterization of SVs remains challenging at the population‐scale. We are optimistic that increased accessibility to long‐read sequencing and advancements in bioinformatic approaches including multireference approaches like genome graphs will alleviate at least some of the challenges associated with resolving SV characteristics below the species level. In the meantime, we address caveats, highlight considerations, and provide recommendations for the characterization of genome‐wide SVs in biodiversity genomic research. [ABSTRACT FROM AUTHOR]

Background: RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. Results: We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Conclusion: Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol. [ABSTRACT FROM AUTHOR]

Increased fructose intake is an international issue. A maternal high-fructose diet during gestation and lactation could affect nervous system development in offspring. Long non-coding RNA (lncRNA) plays an important role in brain biology. However, the mechanism whereby maternal high-fructose diets influence offspring brain development by affecting lncRNAs is still unclear. Here, we administered 13% and 40% fructose water to establish a maternal high-fructose diet model during gestation and lactation. To determine lncRNAs and their target genes, full-length RNA sequencing was performed using the Oxford Nanopore Technologies platform, and 882 lncRNAs were identified. Moreover, the 13% fructose group and the 40% fructose group had differentially expressed lncRNA genes compared with the control group. Enrichment analyses and co-expression analyses were performed to investigate the changes in biological function. Furthermore, enrichment analyses, behavioral science experiments, and molecular biology experiments all indicated that the fructose group offspring showed anxiety-like behaviors. In summary, this study provides insight into the molecular mechanisms underlying maternal high-fructose diet-induced lncRNA expression and co-expression of lncRNA and mRNA. [ABSTRACT FROM AUTHOR]

Most of the current assays directed at the investigation of HIV reactivation are based on cultures of infected cells such as Peripheral Blood Mononuclear Cells (PBMCs) or isolated CD4+ T cells, stimulated in vitro with different activator molecules. The culture media in these in vitro tests lack many age- and donor-specific immunomodulatory components normally found within the autologous plasma. This triggered our interest in understanding the impact that different matrices and cell types have on T cell transcriptional profiles following in vitro culture and stimulation. Methods: Unstimulated or stimulated CD4+ T cells of three young adults with perinatal HIV-infection were isolated from PBMCs before or after culture in RPMI medium or autologous plasma. Transcriptomes were sequenced using Oxford Nanopore technologies. Results: Transcriptional profiles revealed the activation of similar pathways upon stimulation in both media with a higher magnitude of TCR cascade activation in CD4+ lymphocytes cultured in RPMI. Conclusions: These results suggest that for studies aiming at quantifying the magnitude of biological mechanisms under T cell activation, the autologous plasma could better approximate the in vivo environment. Conversely, if the study aims at defining qualitative aspects, then RPMI culture could provide more evident results. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) provide persistent contaminants in freshwaters and the oceans from anthropogenic sources worldwide. MP contamination in ecosystems has emerged as a global environmental issue. While increasing research focused on the ecological consequences of plastic pollution, health-related implications of plastic pollution have been somewhat overlooked. In this study, we evaluated the effects of polyethylene MP contamination on microbial, physical, and biochemical characteristics of the hydrothermal vent crab Xenograpsus testudinatus over a 7-day food exposure. Different concentrations (0%, 0.3%, 0.6%, and 1.0%) of polyethylene MPs were used for feed intake experiments. Oxford Nanopore Technologies’ full-length sequencing of the 16S rDNA gene was used to explore the changes of the microbial composition in vent crab tissues. At the phylum level, the content of Firmicutes significantly decreased in the digestive gland tissue. Furthermore, the predicted functions of genes in the microbial community were significantly influenced by MPs. In contrast, there were eight functions in gill and 11 functions in digestive gland tissues identified at low and high intake levels. The dominant functions of methylotrophy, dark thiosulfate oxidation, dark oxidation of sulfur compounds, aromatic hydrocarbon degradation, and aromatic compound degradation were significantly increased at high intake levels in the digestive gland. These findings indicate that MP ingestion causes not only a short-term decrease in energy intake for crustaceans but also a change in microbial communities and their functions. This study provided a first account on the toxicity of MPs in a hydrothermal vent crab to aid in the assessment of health risks provided by polyethylene MP to marine invertebrates.

BackgroundIn light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies).FindingsUsing a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates.ConclusionsSequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.

Transcriptome studies evaluating whole blood and tissues are often confounded by overrepresentation of highly abundant transcripts. These abundant transcripts are problematic, as they compete with and prevent the detection of rare RNA transcripts, obscuring their biological importance. This issue is more pronounced when using long-read sequencing technologies for isoform-level transcriptome analysis, as they have relatively lower throughput compared to short-read sequencers. As a result, long-read based transcriptome analysis is prohibitively expensive for non-model organisms. While there are off-the-shelf kits available for select model organisms capable of depleting highly abundant transcripts for alpha (HBA) and beta (HBB) hemoglobin, they are unsuitable for non-model organisms. To address this, we have adapted the recent CRISPR/Cas9-based depletion method (depletion of abundant sequences by hybridization) for long-read full-length cDNA sequencing approaches that we call Long-DASH. Using a recombinant Cas9 protein with appropriate guide RNAs, full-length hemoglobin transcripts can be depleted in vitro prior to performing any short- and long-read sequencing library preparations. Using this method, we sequenced depleted full-length cDNA in parallel using both our Oxford Nanopore Technology (ONT) based R2C2 long-read approach, as well as the Illumina short-read based Smart-seq2 approach. To showcase this, we have applied our methods to create an isoform-level transcriptome from whole blood samples derived from three polar bears (Ursus maritimus). Using Long-DASH, we succeeded in depleting hemoglobin transcripts and generated deep Smart-seq2 Illumina datasets and 3.8 million R2C2 full-length cDNA consensus reads. Applying Long-DASH with our isoform identification pipeline, Mandalorion, we discovered ∼6,000 high-confidence isoforms and a number of novel genes. This indicates that there is a high diversity of gene isoforms within U. maritimus not yet reported. This reproducible and straightforward approach has not only improved the polar bear transcriptome annotations but will serve as the foundation for future efforts to investigate transcriptional dynamics within the 19 polar bear subpopulations around the Arctic.

Abstract Objectives Functional genetic variation plays an important role in predicting patients’ response to chemotherapeutic agents. A growing catalogue of mitochondrial DNA (mtDNA) alterations in various cancers point to their important roles in altering the drug responsiveness and survival of cancer cells. In this work, we report the mtDNA sequences, obtained using a nanopore sequencer that can directly sequence unamplified DNA, and the transcriptomes of oral squamous cell carcinoma (OSCC) cell lines with differing responses to cisplatin, to explore the interplay between mtDNA alterations, epigenetic regulation of gene expression, and cisplatin response in OSCC. Data description Two human OSCC cell lines, namely H103 and SAS, and drug-resistant stem-like cells derived from SAS were used in this work. To validate our hypothesis that cisplatin sensitivity is linked to mtDNA changes, we sequenced their mtDNA using a nanopore sequencer, MinION. We also obtained the whole transcriptomic profiles of the cells from a microarray analysis. The mtDNA mutational and whole transcriptomic profiles that we provide can be used alongside other similar datasets to facilitate the identification of new markers of cisplatin sensitivity, and therefore the development of effective therapies for OSCC.

In a previous study, Multiplex-nanopore-sequencing based whole genome sequencing (WGS) allowed for accurate in silico serotype prediction of Salmonella within one day for five multiplexed isolates, using both SISTR and SeqSero2. Since only ten serotypes were tested in our previous study, the conclusions above were yet to be evaluated in a larger scale test. In the current study we evaluated this workflow with 69 Salmonella serotypes and also explored the feasibility of using multiplex-nanopore-sequencing based WGS for antimicrobial resistance gene (AMR) and virulence gene detection. We found that accurate in silico serotype prediction with nanopore-WGS data was achieved within about five hours of sequencing at a minimum of 30× Salmonella genome coverage, with SeqSero2 as the serotype prediction tool. For each tested isolate, small variations were observed between the AMR/virulence gene profiles from the Illumina and Nanopore sequencing platforms. Taking results generated using Illumina data as the benchmark, the average precision value per isolate was 0.99 for both AMR and virulence gene detection. We found that the resistance gene identifier – RGI identified AMR genes with nanopore data at a much lower accuracy compared to Abricate, possibly due to RGI’s less stringent minimum similarity and coverage by default for database matching. This study is an evaluation of multiplex-nanoporesequencing based WGS as a cost-efficient and rapid Salmonella classification method, and a starting point for future validation and verification of using it as a AMR/virulence gene profiling tool for the food industry. This study paves the way for the application of nanopore sequencing in surveillance, tracking, and risk assessment of Salmonella across the food supply chain. [ABSTRACT FROM AUTHOR]

Background: Massive parallel sequencing technologies have enabled the elucidation of plant phylogenetic relationships from chloroplast genomes at a high pace. These include members of the family Rhamnaceae. The current Rhamnaceae phylogenetic tree is from 13 out of 24 Rhamnaceae chloroplast genomes, and only one chloroplast genome of the genus Ventilago is available. Hence, the phylogenetic relationships in Rhamnaceae remain incomplete, and more representative species are needed. Results: The complete chloroplast genome of Ventilago harmandiana Pierre was outlined using a hybrid assembly of long- and short-read technologies. The accuracy and validity of the final genome were confirmed with PCR amplifications and investigation of coverage depth. Sanger sequencing was used to correct for differences in lengths and nucleotide bases between inverted repeats because of the homopolymers. The phylogenetic trees reconstructed using prevalent methods for phylogenetic inference were topologically similar. The clustering based on codon usage was congruent with the molecular phylogenetic tree. The groups of genera in each tribe were in accordance with tribal classification based on molecular markers. We resolved the phylogenetic relationships among six Hovenia species, three Rhamnus species, and two Ventilago species. Our reconstructed tree provides the most complete and reliable low-level taxonomy to date for the family Rhamnaceae. Similar to other higher plants, the RNA editing mostly resulted in converting serine to leucine. Besides, most genes were subjected to purifying selection. Annotation anomalies, including indel calling errors, unaligned open reading frames of the same gene, inconsistent prediction of intergenic regions, and misannotated genes, were identified in the published chloroplast genomes used in this study. These could be a result of the usual imperfections in computational tools, and/or existing errors in reference genomes. Importantly, these are points of concern with regards to utilizing published chloroplast genomes for comparative genomic analysis. Conclusions: In summary, we successfully demonstrated the use of comprehensive genomic data, including DNA and amino acid sequences, to build a reliable and high-resolution phylogenetic tree for the family Rhamnaceae. Additionally, our study indicates that the revision of genome annotation before comparative genomic analyses is necessary to prevent the propagation of errors and complications in downstream analysis and interpretation. [ABSTRACT FROM AUTHOR]

Background: Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT's improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. Results: Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy's plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). Conclusions: The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes. [ABSTRACT FROM AUTHOR]

Abstract Background Structural Variations (SVs) are genomic rearrangements derived from duplication, deletion, insertion, inversion, and translocation events. In the past, SVs detection was limited to cytological approaches, then to Next-Generation Sequencing (NGS) short reads and partitioned assemblies. Nowadays, technologies such as DNA long read sequencing and optical mapping have revolutionized the understanding of SVs in genomes, due to the enhancement of the power of SVs detection. This study aims to investigate performance of two techniques, 1) long-read sequencing obtained with the MinION device (Oxford Nanopore Technologies) and 2) optical mapping obtained with Saphyr device (Bionano Genomics) to detect and characterize SVs in the genomes of the two ecotypes of Arabidopsis thaliana, Columbia-0 (Col-0) and Landsberg erecta 1 (Ler-1). Results We described the SVs detected from the alignment of the best ONT assembly and DLE-1 optical maps of A. thaliana Ler-1 against the public reference genome Col-0 TAIR10.1. After filtering (SV > 1 kb), 1184 and 591 Ler-1 SVs were retained from ONT and Bionano technologies respectively. A total of 948 Ler-1 ONT SVs (80.1%) corresponded to 563 Bionano SVs (95.3%) leading to 563 common locations. The specific locations were scrutinized to assess improvement in SV detection by either technology. The ONT SVs were mostly detected near TE and gene features, and resistance genes seemed particularly impacted. Conclusions Structural variations linked to ONT sequencing error were removed and false positives limited, with high quality Bionano SVs being conserved. When compared with the Col-0 TAIR10.1 reference genome, most of the detected SVs discovered by both technologies were found in the same locations. ONT assembly sequence leads to more specific SVs than Bionano one, the latter being more efficient to characterize large SVs. Even if both technologies are complementary approaches, ONT data appears to be more adapted to large scale populations studies, while Bionano performs better in improving assembly and describing specificity of a genome compared to a reference.