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Introduction: Colonisation and infection with Carbapenem-resistant Enterobacterales (CRE) in healthcare settings poses significant risks, especially for vulnerable patients. Genomic analysis can be used to trace transmission routes, supporting antimicrobial stewardship and informing infection control strategies. Here we used genomic analysis to track the movement and transmission of CREs within clinical and environmental samples. Methods: 25 isolates were cultured from clinical patient samples or swabs, that tested positive for OXA-48-like variants using the NG-Test® CARBA-5 test and whole genome sequenced (WGS) using Oxford Nanopore Technologies (ONT). 158 swabs and 52 wastewater samples were collected from the ward environment. 60 isolates (matching clinical isolate genera; Klebsiella, Enterobacter, Citrobacter and Escherichia) were isolated from the environmental samples using selective agar. Metagenomic sequencing was undertaken on 36 environmental wastewater and swab samples. Results: 21/25 (84%) clinical isolates had > 1 blaOXA gene and 19/25 (76%) harboured > 1 blaNDM gene. Enterobacterales were most commonly isolated from environmental wastewater samples 27/52 (51.9%), then stick swabs 5/43 (11.6%) and sponge swabs 5/115 (4.3%). 11/60 (18%) environmental isolates harboured > 1 blaOXA gene and 1.9% (1/60) harboured blaNDM-1. blaOXA genes were found in 2/36 (5.5%) metagenomic environmental samples. Conclusions: Potential for putative patient-patient and patient-ward transmission was shown. Metagenomic sampling needs optimization to improve sensitivity. [ABSTRACT FROM AUTHOR]

The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches do not allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA + specificity also results in 3′-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30%–50%) of intronic content compared to that of the polyA-specific protocols (5%–8%). In addition, the rRNA depletion protocols enabled ∼20–50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts. [ABSTRACT FROM AUTHOR]

Oxford Nanopore Technology (ONT) allows for the rapid profiling of aquaculture microbiomes. However, not all the experimental and downstream methodological possibilities have been benchmarked. Here, we aimed to offer novel insights into the use of different library preparation methods (standard-RAP and native barcoding-LIG), primers (V3–V4, V1–V3, and V1–V9), and basecalling models (fast-FAST, high-HAC, and super-accuracy-SUP) implemented in ONT to elucidate the microbiota associated with the aquatic environment and farmed fish, including faeces, skin, and intestinal mucus. Microbial DNA from water and faeces samples could be amplified regardless of the library–primer strategy, but only with LIG and V1–V3/V1–V9 primers in the case of skin and intestine mucus. Low taxonomic assignment levels were favoured by the use of full-length V1–V9 primers, though in silico hybridisation revealed a lower number of potential matching sequences in the SILVA database, especially evident with the increase in Actinobacteriota in real datasets. SUP execution allowed for a higher median Phred quality (24) than FAST (11) and HAC (17), but its execution time (6–8 h) was higher in comparison to the other models (0.6–7 h). Altogether, we optimised the use of ONT for water- and fish-related microbial analyses, validating, for the first time, the use of the LIG strategy. We consider that LIG–V1–V9-HAC is the optimal time/cost-effective option to amplify the microbial DNA from environmental samples. However, the use of V1–V3 could help to maximise the dataset microbiome diversity, representing an alternative when long amplicon sequences become compromised by microbial DNA quality and/or high host DNA loads interfere with the PCR amplification/sequencing procedures, especially in the case of gut mucus. [ABSTRACT FROM AUTHOR]

Our goal was to assess the accuracy of next generation sequencing (NGS) compared with Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n = 30) were sequenced with Sanger or reamplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB). For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical (n = 9) or nearly identical (n = 14) compared with Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had ≥35 ambiguous calls in five amplicons and 0 ambiguities in two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template) and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2–13 differences compared with Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared with Sanger and, importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates. [ABSTRACT FROM AUTHOR]

Inflammatory Bowel Disease (IBD), a chronic inflammatory condition affecting the gastrointestinal tract, is associated with extraintestinal manifestations (EIMs) in up to 50% of patients, significantly impacting quality of life and causing serious complications. Reduced gut microbiota bacterial diversity is a characteristic of IBD, and emerging evidence suggests fungal changes, such as an increase in Candida, are also present. However, the mycobiota in patients with IBD, particularly those with EIMs, remains sparsely investigated. Fecal samples from 107 patients with IBD and 43 healthy controls (HC) underwent Internal Transcribed Spacer 2 (ITS2) sequencing using Oxford Nanopore technology. Fecal mycobiota composition differed between patients with IBD and HC, with increased Malassezia in IBD. Patients with IBD and EIMs exhibited lower fungal richness compared to those without EIMs, however did not differ in beta diversity. Patients with IBD and primary sclerosing cholangitis (PSC, n = 7) possessed a distinct mycobiota composition compared with HC and IBD without EIMs, characterized by lower diversity and decreased relative abundance of Saccharomyces cerevisiae and unspecified Dipodascaceae. Overall, the fecal mycobiota of the whole study group (n = 150) exhibits heterogeneity and low richness, primarily shaped by the dominant genus. In conclusion, minor fecal mycobiota differences related to EIMs were observed, particularly in PSCs.

Transposable elements (TEs) increase the frequency of spontaneous mutations in the genome and are capable of altering the gene structure and expression. TE activities and genomic positions are therefore important to study. A combination of two sequencing methods proved advantageous in searching for TE insertions and chromosomal rearrangements, i.e., full-genome nanopore sequencing allowed detection of TE insertions, and transcriptome sequencing on the Illumina platform evaluated their effects on gene expression. Genome sequencing data were obtained for Drosophila melanogaster strains with the SS (w1, flamenco mutant) and MS (w1, flamenco mutant, active gypsy copy) flamenco phenotypes. The wild-type laboratory strain D32 was used as a control. TE insertions and deletions in euchromatin genome regions and gene introns were found in the mutant and wild-type strains as compared with a reference genome (NCBI GCF_000001215.4). The genomes under study were searched for insertions and deletions in RNA interference system genes and genes differentially expressed in the SS and MS strains. TE insertions were detected in various regions of the AGO3, CG17147, Su(var)3-3, Gasz, CG43348, moody, and CG17752 genes. A change in TE position did not correlate with a decrease or an increase in gene transcription in most genes. A chromosomal rearrangement affecting the 3'-untranslated region was observed in the vig gene. A de novo genome assembly was conducted for the MS strain based on the long-read sequencing data. Higher expression of CR45822 and pst in the SS and MS strains was found to be due to a triplication rather than to changes in regulatory sequences or a TE insertion. [ABSTRACT FROM AUTHOR]

Most third-generation sequencing (TGS) processing tools rely on multiple sequence alignment (MSA) methods to manage sequencing errors. Despite the broad range of MSA approaches available, a limited selection of implementations are commonly used in practice for this type of application, and no comprehensive comparative assessment of existing tools has been undertaken to date. In this context, we have developed an automatic pipeline, named MSA Limit, designed to facilitate the execution and evaluation of diverse MSA methods across a spectrum of conditions representative of TGS reads. MSA Limit offers insights into alignment accuracy, time efficiency, and memory utilization. It serves as a valuable resource for both users and developers, aiding in the assessment of algorithmic performance and assisting users in selecting the most appropriate tool for their specific experimental settings. Through a series of experiments using real and simulated data, we demonstrate the value of such exploration. Our findings reveal that in certain scenarios, popular methods may not consistently exhibit optimal efficiency and that the choice of the most effective method varies depending on factors such as sequencing depth, genome characteristics, and read error patterns. MSA Limit is an open source and freely available tool. All code and data pertaining to it and this manuscript are available at . [ABSTRACT FROM AUTHOR]

ABSTRACT Highly pathogenic avian influenza viruses continue to pose global risks to One Health, including agriculture, public, and animal health. Rapid and accurate genomic surveillance is critical for monitoring viral mutations, tracing transmission, and guiding interventions in near real-time. Oxford Nanopore sequencing holds promise for real-time influenza genotyping, but data quality from R9 chemistry has limited its adoption due to challenges resolving low-complexity regions such as the biologically critical hemagglutinin cleavage site, a homopolymer of basic amino acids that distinguish highly pathogenic strains. In this study, human and avian influenza isolates (n = 45) from Cambodia were sequenced using both R9.4.1 and R10.4.1 flow cells and chemistries to evaluate performance between approaches. Overall, R10.4.1 yielded increased data output with higher average quality compared to R9.4.1, producing improved consensus sequences using a reference-based bioinformatics approach. R10.4.1 had significantly lower minor population insertion and deletion frequencies, driven by improved performance in low sequence complexity regions prone to insertion and deletion errors, such as homopolymers. Within the hemagglutinin cleavage site, R10.4.1 resolved the correct motif in 90% of genomes compared to only 60% with R9.4.1. Further examination showed reduced frameshift mutations in consensus sequences generated with R10.4.1 that could result in incorrectly classified virulence with automated pipelines. Improved consensus genome quality from nanopore sequencing approaches, especially across biologically important low-complexity regions, is critical to reduce subjective hand-curation and will improve local and global genomic surveillance responses.IMPORTANCEThis study demonstrates significant advancement in the field of influenza virus genomic surveillance by showcasing the superior accuracy and data quality of the Oxford Nanopore R10 sequencing chemistry compared to the older R9 chemistry. Improved resolution, including in the critical hemagglutinin multi-basic cleavage site, enables more reliable monitoring and tracking of viral mutations. This accelerates the ability to respond quickly to outbreaks, potentially improving impacts on public health, agriculture, and the economy by enabling more accurate and timely interventions.

The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches do not allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA + specificity also results in 3′-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30%–50%) of intronic content compared to that of the polyA-specific protocols (5%–8%). In addition, the rRNA depletion protocols enabled ∼20–50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts.

Simple Summary: Understanding the genetic makeup of microorganisms provides information on their evolutionary relationship with other similar organisms. It also sheds light on their ability to cause disease in susceptible individuals and to resist the hosts' defense mechanisms, including the effects of antimicrobial therapy. In this study, we performed complete genome sequencing and characterization of bacteria called Arcanobacterium hippocoleae, which were isolated from the reproductive tract of infected mares. These bacteria displayed small and large phenotypes when grown on artificial media. The whole genome sequences between the large phenotypes were closely related, while the small and large types were distant from each other in terms of sequence size and identity. Putative genetic elements that might be associated with disease conditions were identified in both bacterial phenotypes. Several genes that express hypothetical proteins with unknown functions were also detected. This study provides the complete genetic structure and analysis of different phenotypes of Arcanobacterium hippocoleae and will serve as a benchmark for future studies to identify the potential function and role of the bacterial proteins in the ability of the bacterium to produce reproductive tract diseases in mares. Arcanobacterium hippocoleae is a Gram-positive fastidious bacterium and is occasionally isolated from the reproductive tract of apparently healthy mares (Equus caballus) or from mares with reproductive tract abnormalities. Apart from a few 16S rRNA gene-based GenBank sequences and one recent report on complete genome assembly, detailed genomic sequence and clinical experimental data are not available on the bacterium. Recently, we observed an unusual increase in the detection of the organism from samples associated with mare reproductive failures in Atlantic Canada. Two colony morphotypes (i.e., small, and large) were detected in culture media, which were identified as A. hippocoleae by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Here, we report the whole genome sequencing and characterization of the morphotype variants. The genome length of the large phenotypes was between 2.42 and 2.43, and the small phenotype was 1.99 Mbs. The orthologous nucleotide identity between the large colony phenotypes was ~99%, and the large and small colony phenotypes was between 77.86 and 78.52%, which may warrant the classification of the two morphotypes into different species. Phylogenetic analysis based on 16S rRNA genes or concatenated housekeeping genes grouped the small and large colony variants into two different genotypic clusters. The UvrA protein, which is part of the nucleotide excision repair (NER) system, and 3-isopropoylmalate dehydratase small subunit protein expressed by the leuD gene were identified as potential virulence factors in the large and small colony morphotypes, respectively. However, detailed functional studies will be required to determine the exact roles of these and other identified hypothetical proteins in the cellular metabolism and potential pathogenicity of A. hippocoleae in mares. [ABSTRACT FROM AUTHOR]

ABSTRACT: Avian pathogenic Escherichia coli (APEC) cause avian colibacillosis and accurately distinguishing infectious isolates is critical for controlling its transmission. Multilocus sequence typing (MLST) is an accurate and efficient strain identification method for epidemiological surveillance. This research aimed to develop a fast and high-throughput workflow that simultaneously sequences the Achtman typing scheme's 7 housekeeping genes of multiple E. coli isolates using the Oxford Nanopore Technologies (ONT) platform for large-scale APEC study. E. coli strains were isolated from poultry farms, the housekeeping genes were amplified, and amplicons were sequenced on an R9.4 MinION flow cell using the Nanopore GridION sequencer (ONT, Oxford, UK) following the initial workflow (ONT-MLST). Moreover, the workflow was revised by introducing large-scale DNA extraction and multiplex PCR into the ONT-MLST workflow and applied to 242 new isolates, 18 isolates from the previous workflow, and 5 ATCC reference strains using Flongle flow cell on the Nanopore MinION Mk1C sequencer (ONT, Oxford, UK). Finally, the sequence type (ST) results of the 308 isolates collected from infected chickens and poultry farm environments were reported and analyzed. Data indicated that E. coli belonging to ST159, ST8578, and ST355 have the potential to infect multiple organs in broiler. In addition, zoonotic STs, ST69, ST10, ST38, and ST131, were detected from poultry farms. With the advantages of the high throughput of ONT, this study provides a rapid workflow for large-scale E. coli typing and identified frequently isolated sequence types related to APEC infection in poultry.

Most third-generation sequencing (TGS) processing tools rely on multiple sequence alignment (MSA) methods to manage sequencing errors. Despite the broad range of MSA approaches available, a limited selection of implementations are commonly used in practice for this type of application, and no comprehensive comparative assessment of existing tools has been undertaken to date. In this context, we have developed an automatic pipeline, named MSA Limit, designed to facilitate the execution and evaluation of diverse MSA methods across a spectrum of conditions representative of TGS reads. MSA Limit offers insights into alignment accuracy, time efficiency, and memory utilization. It serves as a valuable resource for both users and developers, aiding in the assessment of algorithmic performance and assisting users in selecting the most appropriate tool for their specific experimental settings. Through a series of experiments using real and simulated data, we demonstrate the value of such exploration. Our findings reveal that in certain scenarios, popular methods may not consistently exhibit optimal efficiency and that the choice of the most effective method varies depending on factors such as sequencing depth, genome characteristics, and read error patterns. MSA Limit is an open source and freely available tool. All code and data pertaining to it and this manuscript are available at https://gitlab.cristal.univ-lille.fr/crohmer/msa-limit.

ABSTRACT European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (–, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB – colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB.IMPORTANCEThis study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

BackgroundGenome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.ResultsHere, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.ConclusionsOur work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.

The aim of the study was to assemble full-length nucleotide sequences of the chromosome and plasmids for 13 Yersinia pestis strains from 11 natural plague foci located in the Russian Federation, using data from two sequencing technologies.Materials and methods. Y. pestis strains were grown on Hottinger’s agar (pH 7.2) at 37 °C. DNA was isolated using phenol-chloroform extraction. For the MinIon genetic analyzer (Oxford Nanopore), DNA fragments were prepared by ligation according to a modified protocol. For the Ion S5 genetic analyzer (IonTorrent), sample preparation was carried out according to the standard protocol for obtaining a library with a DNA fragment size of 400 nucleotide pairs (bp). The resulting single reads were filtered by average quality Q30 for IonTorrent and Q7 for Oxford Nanopore.Results and discussion. DNA fragments containing 50 000 or more nucleotide pairs were prepared for subsequent sequencing using nanopore sequencing technology (Oxford Nanopore). The Trycycler algorithm was applied for hybrid assembly of the genome of Y. pestis strains and correction of errors arising during this process, allowing the obtainment of full-length nucleotide sequences of the chromosome and plasmids for each genome of the strain. The nucleotide sequences of the chromosomes of 13 Y. pestis strains from 11 natural foci of plague located in the Russian Federation have been deposited in the international genetic database NCBI GenBank. It has been established that to assemble full-length genomes of Y. pestis strains, a significant number of reads with a size of 50 000 bp or more is required, and the use of the Trycycler algorithm allows one to generate a more accurate assembly of complete bacterial genomes.

Introduction: African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional "readthrough" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3' RNA-seq indicates an accumulation of mRNA 3' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. Methods: Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3' RNA-seq with 3' ends mapped by LRS during early and late infection. Results: Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors.Our results cement this T-richmotif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. Discussion: This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection. [ABSTRACT FROM AUTHOR]

Septoria brown spot, caused by Septoria glycines, is Illinois’ most prevalent soybean disease. It is common to use foliar fungicides to control Septoria brown spot and other late-season diseases of soybean. The effects of fungicide on nontarget organisms in the phyllosphere are unknown. To study the effect of S. glycines and fungicide application on the soybean phyllosphere mycobiome, we conducted a replicated field trial and collected samples at three soybean developmental stages. Then, we sequenced full-length internal transcribed spacer and a partial large subunit region using Oxford Nanopore technologies. Sequencing and data analysis produced 3,342 operational taxonomic units. The richness of the fungal community increased with the host development. There were differences in mycobiome diversity between soybean lines at the early developmental stage but not at the reproductive stages. Inoculation with S. glycines did not affect the α diversity but some significant changes were observed for the β diversity. At the beginning seed stage (R5), fungicide application changed the composition of the fungal community. The fungicide treatment decreased the proportion of several fungal taxa but it increased the proportion of Septoria. The core mycobiome in the phyllosphere was composed of genera Gibberella, Alternaria, Didymella, Cladosporium, Plectosphaerella, Colletotrichum, and Bipolaris. Network analysis identified significant interactions between Septoria and Diaporthe, Bipolaris, and two other taxonomic units. In this study, we set Septoria as the target organism and demonstrated that metabarcoding could be a tool to quantify the effect of multiple treatments on the mycobiome community. Better understanding of the dynamics of the phyllosphere microbiome is necessary to untangle the late-season diseases of soybean.

ABSTRACTThe genus Vibrio includes pathogenic bacteria able to cause disease in humans and aquatic organisms, leading to disease outbreaks and significant economic losses in the fishery industry. Despite much work on Vibrio in several marine organisms, no specific studies have been conducted on Anadara tuberculosa. This is a commercially important bivalve species, known as “piangua hembra,” along Colombia’s Pacific coast. Therefore, this study aimed to identify and characterize the genomes of Vibrio isolates obtained from A. tuberculosa. Bacterial isolates were obtained from 14 A. tuberculosa specimens collected from two locations along the Colombian Pacific coast, of which 17 strains were identified as Vibrio: V. parahaemolyticus (n = 12), V. alginolyticus (n = 3), V. fluvialis (n = 1), and V. natriegens (n = 1). Whole genome sequence of these isolates was done using Oxford Nanopore Technologies (ONT). The analysis revealed the presence of genes conferring resistance to β-lactams, tetracyclines, chloramphenicol, and macrolides, indicating potential resistance to these antimicrobial agents. Genes associated with virulence were also found, suggesting the potential pathogenicity of these Vibrio isolates, as well as genes for Type III Secretion Systems (T3SS) and Type VI Secretion Systems (T6SS), which play crucial roles in delivering virulence factors and in interbacterial competition. This study represents the first genomic analysis of bacteria within A. tuberculosa, shedding light on Vibrio genetic factors and contributing to a comprehensive understanding of the pathogenic potential of these Vibrio isolates.IMPORTANCEThis study presents the first comprehensive report on the whole genome analysis of Vibrio isolates obtained from Anadara tuberculosa, a bivalve species of great significance for social and economic matters on the Pacific coast of Colombia. Research findings have significant implications for the field, as they provide crucial information on the genetic factors and possible pathogenicity of Vibrio isolates associated with A. tuberculosa. The identification of antimicrobial resistance genes and virulence factors within these isolates emphasizes the potential risks they pose to both human and animal health. Furthermore, the presence of genes associated with Type III and Type VI Secretion Systems suggests their critical role in virulence and interbacterial competition. Understanding the genetic factors that contribute to Vibrio bacterial virulence and survival strategies within their ecological niche is of utmost importance for the effective prevention and management of diseases in aquaculture practices.

Forest soil and leaf litter support diverse arthropod mesofauna for which diversity dynamics is challenging to study due to the high number of species and specimens, small body size and limited taxonomic knowledge.Immature stages (larvae) are even harder to identify than adults, as their morphology is largely unknown. Therefore, larvae are often ignored, even though they may form a substantial proportion of specimens collected, are less mobile than adults and their exclusion may provide an incomplete diversity profile.Here, we use Oxford Nanopore DNA barcoding to investigate whether the inclusion of larvae provides a more complete taxonomic, phylogenetic and functional diversity profiles in leaf litter beetles (Coleoptera) from a subtropical forest in Taiwan.Our results indicate that larvae represent up to 38% of beetle specimens per sample, but most of them belong to 2–3 common species. Larvae of most beetle species are rarely collected repeatedly or in multiple specimens, possibly due to special habitat requirements or high seasonality.Taxonomic, phylogenetic and incidence‐based functional diversity measures were not affected by the exclusion of larvae in the Staphylinidae dataset but were strongly biased in all‐beetle dataset, especially when only common or abundant species were considered. Taxonomic beta diversity was not affected by the omission of larvae.Our results indicate that immature stages may be omitted in ecological studies of arthropods in case both adults and larvae co‐occur in the same habitat, and the sites are sampled repeatedly. Caution is needed (1) in groups in which larvae and adults do not inhabit the same environment or strongly differ in biology, (2) when rare species are omitted or (3) when functional diversity is calculated from abundance data. [ABSTRACT FROM AUTHOR]

Sour cherry (Prunus cerasus L.) is an important allotetraploid cherry species that evolved in the Caspian Sea and Black Sea regions from a hybridization of the tetraploid ground cherry (Prunus fruticosa Pall.) and an unreduced pollen of the diploid sweet cherry (P. avium L.) ancestor. Details of when and where the evolution of this species occurred are unclear, as well as the effect of hybridization on the genome structure. To gain insight, the genome of the sour cherry cultivar 'Schattenmorelle' was sequenced using Illumina NovaSeqTM and Oxford Nanopore long-read technologies, resulting in a ~629-Mbp pseudomolecule reference genome. The genome could be separated into two subgenomes, with subgenome PceS_a originating from P. avium and subgenome PceS_f originating from P. fruticosa. The genome also showed size reduction compared to ancestral species and traces of homoeologous sequence exchanges throughout. Comparative analysis confirmed that the genome of sour cherry is segmental allotetraploid and evolved very recently in the past. [ABSTRACT FROM AUTHOR]

Recent advances in long-read sequencing have enabled the creation of reference-quality genome assemblies for multiple individuals within a species. In particular, 8 long-read genome assemblies have recently been published for the canine model (dogs and wolves). These assemblies were created using a range of sequencing and computational approaches, with only limited comparisons described among subsets of the assemblies. Here we present 3 high-quality de novo reference assemblies based upon Oxford Nanopore long-read sequencing: 2 Bernese Mountain Dogs (BD & OD) and a Cairn terrier (CA611). These breeds are of particular interest due to the enrichment of unresolved genetic disorders. Leveraging advancement in software technologies, we utilized published data of Labrador Retriever (Yella) to generate a new assembly, resulting in a ~280-fold increase in continuity (N50 size of 91 kbp vs 25.75 Mbp). In conjunction with these 4 new assemblies, we uniformly assessed 8 existing assemblies for generalized quality metrics, sequence divergence, and a detailed BUSCO assessment. We identified a set of ~400 conserved genes during the BUSCO analysis missing in all assemblies. Genome-wide methylation profiles were generated from the nanopore sequencing, resulting in broad concordance with existing wholegenome and reduced-representation bisulfite sequencing, while highlighting superior overage of mobile elements. These analyses demonstrate the ability of Nanopore sequencing to resolve the sequence and epigenetic profile of canine genomes. [ABSTRACT FROM AUTHOR]

The first insect genome assembly (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 insect species representing 20 orders. In this study, we analyzed the most-contiguous assembly for each species and provide a "state-of-the-field" perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technologies. Relative to species richness, genomic efforts have been biased toward four orders (Diptera, Hymenoptera, Collembola, and Phasmatodea), Coleoptera are underrepresented, and 11 orders still lack a publicly available genome assembly. The average insect genome assembly is 439.2 Mb in length with 87.5% of single-copy benchmarking genes intact. Most notable has been the impact of long-read sequencing; assemblies that incorporate long reads are ∼48× more contiguous than those that do not. We offer four recommendations as we collectively continue building insect genome resources: 1) seek better integration between independent research groups and consortia, 2) balance future sampling between filling taxonomic gaps and generating data for targeted questions, 3) take advantage of long-read sequencing technologies, and 4) expand and improve gene annotations.

The Andean bear is the only extant member of the Tremarctine subfamily and the only extant ursid species to inhabit South America. Here, we present an annotated de novo assembly of a nuclear genome from a captive-born female Andean bear, Mischief, generated using a combination of short and long DNA and RNA reads. Our final assembly has a length of 2.23 Gb, and a scaffold N50 of 21.12 Mb, contig N50 of 23.5 kb, and BUSCO score of 88%. The Andean bear genome will be a useful resource for exploring the complex phylogenetic history of extinct and extant bear species and for future population genetics studies of Andean bears.

Arcanobacterium hippocoleae is a Gram-positive fastidious bacterium and is occasionally isolated from the reproductive tract of apparently healthy mares (Equus caballus) or from mares with reproductive tract abnormalities. Apart from a few 16S rRNA gene-based GenBank sequences and one recent report on complete genome assembly, detailed genomic sequence and clinical experimental data are not available on the bacterium. Recently, we observed an unusual increase in the detection of the organism from samples associated with mare reproductive failures in Atlantic Canada. Two colony morphotypes (i.e., small, and large) were detected in culture media, which were identified as A. hippocoleae by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Here, we report the whole genome sequencing and characterization of the morphotype variants. The genome length of the large phenotypes was between 2.42 and 2.43, and the small phenotype was 1.99 Mbs. The orthologous nucleotide identity between the large colony phenotypes was ~99%, and the large and small colony phenotypes was between 77.86 and 78.52%, which may warrant the classification of the two morphotypes into different species. Phylogenetic analysis based on 16S rRNA genes or concatenated housekeeping genes grouped the small and large colony variants into two different genotypic clusters. The UvrA protein, which is part of the nucleotide excision repair (NER) system, and 3-isopropoylmalate dehydratase small subunit protein expressed by the leuD gene were identified as potential virulence factors in the large and small colony morphotypes, respectively. However, detailed functional studies will be required to determine the exact roles of these and other identified hypothetical proteins in the cellular metabolism and potential pathogenicity of A. hippocoleae in mares.

Sour cherry (Prunus cerasus L.) is an important allotetraploid cherry species that evolved in the Caspian Sea and Black Sea regions from a hybridization of the tetraploid ground cherry (Prunus fruticosa Pall.) and an unreduced pollen of the diploid sweet cherry (P. avium L.) ancestor. Details of when and where the evolution of this species occurred are unclear, as well as the effect of hybridization on the genome structure. To gain insight, the genome of the sour cherry cultivar ‘Schattenmorelle’ was sequenced using Illumina NovaSeqTM and Oxford Nanopore long-read technologies, resulting in a ~629-Mbp pseudomolecule reference genome. The genome could be separated into two subgenomes, with subgenome PceS_a originating from P. avium and subgenome PceS_f originating from P. fruticosa. The genome also showed size reduction compared to ancestral species and traces of homoeologous sequence exchanges throughout. Comparative analysis confirmed that the genome of sour cherry is segmental allotetraploid and evolved very recently in the past.

Ascidians have the potential to reveal fundamental biological insights related to coloniality, regeneration, immune function, and the evolution of these traits. This study implements a hybrid assembly technique to produce a genome assembly and annotation for the botryllid ascidian, Botrylloides violaceus. A hybrid genome assembly was produced using Illumina, Inc. short and Oxford Nanopore Technologies long-read sequencing technologies. The resulting assembly is comprised of 831 contigs, has a total length of 121 Mbp, N50 of 1 Mbp, and a BUSCO score of 96.1%. Genome annotation identified 13 K protein-coding genes. Comparative genomic analysis with other tunicates reveals patterns of conservation and divergence within orthologous gene families even among closely related species. Characterization of the Wnt gene family, encoding signaling ligands involved in development and regeneration, reveals conserved patterns of subfamily presence and gene copy number among botryllids. This supports the use of genomic data from nonmodel organisms in the investigation of biological phenomena. [ABSTRACT FROM AUTHOR]

Whole-genome sequencing data from wild-caught individuals of closely related North American killifish species (Fundulus xenicus, Fundulus catenatus, Fundulus nottii, and Fundulus olivaceus) were obtained using long-read Oxford Nanopore Technology (ONT) PromethION and short-read Illumina platforms. Draft de novo reference genome assemblies were generated using a combination of long and short sequencing reads. For each species, the PromethION platform was used to generate 30-45× sequence coverage, and the Illumina platform was used to generate 50-160× sequence coverage. Illumina-only assemblies were fragmented with high numbers of contigs, while ONT-only assemblies were error prone with low BUSCO scores. The highest N50 values, ranging from 0.4 to 2.7 Mb, were from assemblies generated using a combination of short- and long-read data. BUSCO scores were consistently >90% complete using the Eukaryota database. High-quality genomes can be obtained from a combination of using short-read Illumina data to polish assemblies generated with long-read ONT data. Draft assemblies and raw sequencing data are available for public use. We encourage use and reuse of these data for assembly benchmarking and other analyses.

Abstract Background Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. Results HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. Conclusions Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

Members of the fish family Poeciliidae (livebearing ‘tooth-carps’) have historically been used as models in medical research, behavior ecology, and biological control. This group of primarily freshwater fishes is highly tolerant to environmental factors such as salinity and warm temperatures and includes some invasive species. Here, we present the mitochondrial genome of Poecilia parae. A representative of this species was obtained from Suriname. The complete mitochondrial genome was sequenced using Oxford Nanopore technology and is 16,559 bp long. The genome contains 13 protein-coding genes, two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and one control region (D-loop). Phylogenetic analysis yielded topologies similar to those previously published. The data generated here will be useful in future studies of comparative biology and those utilizing environmental DNA (eDNA).

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both shortread NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA. [ABSTRACT FROM AUTHOR]

Simple Summary: Detection and genotyping of genetic variants across genomes has several applications that include, e.g., identification of genetic background of phenotypic traits, detection of disease-related variation, and analysis of population genetic structure. In this study, we attempt to develop a Nanopore sequencing-based genotyping strategy (with MinION system from Oxford Nanopore) that allows simple and cost-efficient genome-wide analysis in horse species. With this method, we generated 28,426 polymorphisms that were genotyped with high accuracy, with a level of error not exceeding 3%. The method can be further improved to increase the number of detected variants and improve their reliability by increasing the sequencing depth. Nanopore sequencing is a third-generation biopolymer sequencing technique that relies on monitoring the changes in an electrical current that occur as nucleic acids are passed through a protein nanopore. Increasing quality of reads generated by nanopore sequencing systems encourages their application in genome-wide polymorphism detection and genotyping. In this study, we employed nanopore sequencing to identify genome-wide polymorphisms in the horse genome. To reduce the size and complexity of genome fragments for sequencing in a simple and cost-efficient manner, we amplified random DNA fragments using a modified DOP-PCR and sequenced the resulting products using the MinION system. After initial filtering, this generated 28,426 polymorphisms, which were validated at a 3% error rate. Upon further filtering for polymorphism and reproducibility, we identified 9495 SNPs that reflected the horse population structure. To conclude, the use of nanopore sequencing, in conjunction with a genome enrichment step, is a promising tool that can be practical in a variety of applications, including genotyping, population genomics, association studies, linkage mapping, and potentially genomic selection. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a vascular plant pathogenic bacterium native to the Americas that is causing significant epidemics and economic losses in olive and almonds in Europe, where it is a quarantine pathogen. Since its first detection in 2013 in Italy, mandatory surveys across Europe revealed the presence of the bacterium also in France, Spain, and Portugal. Combining Oxford Nanopore Technologies and Illumina sequencing data, we assembled high-quality complete genomes of seven X. fastidiosa subsp. fastidiosa strains isolated from different plants in Spain, the United States, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using the Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmids pXF-P1.CFBP8073 and pXF-P2.CFBP8073) not previously described in X. fastidiosa and one (pXF5823.CFBP8073) almost identical to a plasmid described in a X. fastidiosa strain from citrus. Plasmids found in the Spanish strains here were similar to those described previously in other strains from the same subspecies and ST1 isolated in the Balearic Islands and the United States. The genome resources from this work will assist in further studies on the role of plasmids in the epidemiology, ecology, and evolution of this plant pathogen. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) is a major rice pathogen, and its genome harbors extensive inter-strain and inter-lineage variations. The emergence of highly virulent pathotypes of Xoo that can overcome major resistance (R) genes deployed in rice breeding programs is a grave threat to rice cultivation. The present study reports on a long-read Oxford nanopore-based complete genomic investigation of Xoo isolates from 11 pathotypes that are reported based on their reaction toward 10 R genes. The investigation revealed remarkable variation in the genome structure in the strains belonging to different pathotypes. Furthermore, transcription activator-like effector (TALE) proteins secreted by the type III secretion system display marked variation in content, genomic location, classes, and DNA-binding domain. We also found the association of tal genes in the vicinity of regions with genome structural variations. Furthermore, in silico analysis of the genome-wide rice targets of TALEs allowed us to understand the emergence of pathotypes compatible with major R genes. Long-read, cost-effective sequencing technologies such as nanopore can be a game changer in the surveillance of major and emerging pathotypes. The resource and findings will be invaluable in the management of Xoo and in appropriate deployment of R genes in rice breeding programs. [ABSTRACT FROM AUTHOR]

Premise: We present approaches used to generate long‐read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long‐read sequencing data determine which steps may be necessary for optimizing output and results. Methods: Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide‐bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA. Results: Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced. Discussion: Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly. [ABSTRACT FROM AUTHOR]

BackgroundLong read sequencing technologies such as Oxford Nanopore can greatly decrease the complexity of de novo genome assembly and large structural variation identification. Currently Nanopore reads have high error rates, and the errors often cluster into low-quality segments within the reads. The limited sensitivity of existing read-based error correction methods can cause large-scale mis-assemblies in the assembled genomes, motivating further innovation in this area.ResultsHere we developed a Convolutional Neural Network (CNN) based method, called MiniScrub, for identification and subsequent "scrubbing" (removal) of low-quality Nanopore read segments to minimize their interference in downstream assembly process. MiniScrub first generates read-to-read overlaps via MiniMap2, then encodes the overlaps into images, and finally builds CNN models to predict low-quality segments. Applying MiniScrub to real world control datasets under several different parameters, we show that it robustly improves read quality, and improves read error correction in the metagenome setting. Compared to raw reads, de novo genome assembly with scrubbed reads produces many fewer mis-assemblies and large indel errors.ConclusionsMiniScrub is able to robustly improve read quality of Oxford Nanopore reads, especially in the metagenome setting, making it useful for downstream applications such as de novo assembly. We propose MiniScrub as a tool for preprocessing Nanopore reads for downstream analyses. MiniScrub is open-source software and is available at https://bitbucket.org/berkeleylab/jgi-miniscrub .

Nocardia species do not replicate as rapidly as other pyogenic bacteria and nocardial infections can be highly fatal, particularly in immunocompromised patients. Here, we present the first report of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis diagnosed by next-generation sequencing (NGS) using the Oxford Nanopore Technologies' MinION device. The bacterium was not identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Due to its low equipment cost, short turn-around-time, and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both short-read NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA.

Abstract Premise We present approaches used to generate long‐read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long‐read sequencing data determine which steps may be necessary for optimizing output and results. Methods Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide‐bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA. Results Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced. Discussion Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.

Background: Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. Results: HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. Conclusions: Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies. [ABSTRACT FROM AUTHOR]

Despite increasing sequencing efforts, numerous fish families still lack a reference genome, which complicates genetic research. One such understudied family is the sand lances (Ammodytidae, literally: "sand burrower"), a globally distributed clade of over 30 fish species that tend to avoid tidal currents by burrowing into the sand. Here, we present the first annotated chromosome-level genome assembly of the great sand eel (Hyperoplus lanceolatus). The genome assembly was generated using Oxford Nanopore Technologies long sequencing reads and Illumina short reads for polishing. The final assembly has a total length of 808.5 Mbp, of which 97.1% were anchored into 24 chromosome-scale scaffolds using proximity-ligation scaffolding. It is highly contiguous with a scaffold and contig N50 of 33.7 and 31.3 Mbp, respectively, and has a BUSCO completeness score of 96.9%. The presented genome assembly is a valuable resource for future studies of sand lances, as this family is of great ecological and commercial importance and may also contribute to studies aiming to resolve the suprafamiliar taxonomy of bony fishes. [ABSTRACT FROM AUTHOR]

The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer's disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? SUMMARY ANSWER Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. WHAT IS KNOWN ALREADY Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. STUDY DESIGN, SIZE, DURATION This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5–10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. MAIN RESULTS AND THE ROLE OF CHANCE The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. LIMITATIONS, REASONS FOR CAUTION The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. WIDER IMPLICATIONS OF THE FINDINGS Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]