Illumina Short Reads Research Articles

A case of penicillin-resistant group B Streptococcus isolated from a patient in the UK.

In England, group B streptococci (GBS; Streptococcus agalactiae) are considered universally susceptible to penicillin. Reports from Africa, Asia, North America and a few European countries have described GBS isolates with penicillin MICs above the epidemiological cut-off (0.125 mg/L). Our aim was to characterize a penicillin-resistant GBS (PRGBS) isolate recovered in 2016 from a patient treated with long-term antimicrobials in the UK. Antibiotic susceptibility of a referred isolate from a discharging sinus overlying a chronic prosthetic joint infection was determined using gradient strip testing for seven antibiotics. Illumina short read sequencing was carried out using a HiSeq 2500 platform to determine MLST, capsular type, to detect mutations in the pbp genes, and to compare the isolate with contemporaneous GBS isolates circulating in the UK. The GBS isolate belonged to capsular type Ia and MLST 144. We observed resistance to penicillin (MIC = 1 mg/L) and tetracycline (32 mg/L) with susceptibility to linezolid (1 mg/L), erythromycin (0.064 mg/L), clindamycin (0.064 mg/L), teicoplanin (0.064 mg/L) and vancomycin (0.25 mg/L). Deduced amino acid sequences revealed substitutions and non-synonymous changes in PBP2x and PBP2b. Genomic analysis of contemporaneous cases (n = 34) from across the UK identified single nucleotide polymorphism (SNP) variation ranged from 153-6596 SNPs. We confirm the first identification of a PRGBS isolate amongst referrals to the UK's national reference laboratory. Substitutions in pbp1a, pbp2a, pbp2x and pbp2b were identified that likely developed in the face of long-term beta-lactam antibiotic use.

Improved resolution of microbial diversity in deep-sea surface sediments using PacBio long-read 16S rRNA gene sequencing.

The PacBio long-read platform, with its exceptional base-level resolution exceeding 99%, has advanced our comprehension of deep-sea microbial diversity. By comparing microbial community analyses conducted using the Illumina short-read and PacBio long-read sequencing platforms, we have provided an enhanced understanding of fine spatial-scale patterns in microbial community diversity with depth across a deep-sea sediment core, as well as methodological insights that will be valuable for future research in this field.

Genome sequencing and transcriptome analysis provide an insight into the hypoxia resistance of Channa asiatica

Channa asiatica is an economically valuable fish species and excellent model for studying hypoxic tolerance. However, the underlying genetic and molecular mechanisms are poorly understood. In this study, we assembled a high-quality C. asiatica genome (23 chromosomes, totaling 722 Mb) using a combination of Illumina short-read, PacBio long-read, and Hi-C sequencing. Repetitive elements accounted for 28.39%of the C. asiatica genome, and 23,949 protein-coding genes were predicted, with 96.63 % of these functionally annotated. Moreover, a comparative genomic analysis of 12 fish genomes showed that gene families associated with oxygen binding and transport were expanded in C. asiatica. In addition, transcriptome analysis revealed that multiple oxidative stress pathways were activated when C. asiatica was exposed to air. In conclusion, this study provided high-quality genome assembly and transcriptome data, both serving as critical resources for researching the genetic basis of hypoxic tolerance in C. asiatica.

Full-length transcriptome assembly of black amur bream (Megalobrama terminalis) as a reference resource.

The genus Megalobrama holds significant economic value in China, with M. terminalis (Black Amur bream) ranking second in production within this group. However, lacking comprehensive genomic and transcriptomic data has impeded research progress. This study aims to fill this gap through an extensive transcriptomic analysis of M. terminalis. We utilized PacBio Isoform Sequencing to generate 558,998 subreads, totaling 45.52 Gb, which yielded 22,141 transcripts after rigorous filtering and clustering. Complementary Illumina short-read sequencing corrected 967,114 errors across these transcripts. Our analysis identified 12,426 non-redundant isoforms, with 11,872 annotated in various databases. Functional annotation indicated 11,841 isoforms matched entries in the NCBI non-redundant protein sequences database. Gene Ontology analysis categorized 10,593 isoforms, revealing strong associations with cellular processes and binding functions. Additionally, 8203 isoforms were mapped to pathways in the Kyoto Encyclopedia of Genes and Genomes, highlighting significant involvement in immune system processes and complement cascades. We notably identified key immune molecules such as alpha-2-macroglobulin and complement component 3, each with multiple isoforms, underscoring their potential roles in the immune response. Our analysis also uncovered 853 alternative splicing events, predominantly involving retained introns, along with 672 transcription factors and 426 long non-coding RNAs. The high-quality reference transcriptome generated in this study provides a valuable resource for comparative genomic studies within the Megalobrama genus, supporting future research to enhance aquaculture stocks.

A chromosome-scale genome assembly of the peanut beetle, Ulomoides dermestoides (Coleptera: Tenebrionidae)

Ulomoides dermestoides, a member of the Tenebrionidae family, is known by various common names such as Chinese beetle, Chinese weevil, peanut beetle, cancer beetle, or asthma beetle. Despite its origin in Asia, it has achieved global distribution owing to its perceived medicinal properties. In this study, we employed a combination of Illumina short-read, PacBio long-read, and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to produce a high-quality, chromosome-scale genome assembly of U. dermestoides. The assembled genome spans 253.38 Mb across 11 chromosomes, including the two sex chromosomes, X and Y. Key metrics include contig and scaffold N50 values of 9.83 Mb and 28.62 Mb, respectively, with an estimated genome completeness of 99.62%. Annotation efforts identified 15,553 protein-coding genes and revealed that 48.11% of the genome comprises repetitive sequences. This meticulously assembled genome serves as a pivotal resource for elucidating the genetic determinants underlying the species’ ecological habits and potential medicinal properties.

Chromosome genome assembly and annotation of Adzuki Bean (Vigna angularis)

Adzuki bean (Vigna angularis) is a significant dietary legume crop that is prevalent in East Asia. It also holds traditional medicinal importance in China. In this study, we report a high-quality, chromosome-level genome assembly of adzuki bean obtained by employing Illumina short-read sequencing, PacBio long-read sequencing, and Hi-C technology. The assembly spans 447.8 Mb, encompassing 96.32% of the estimated genome, with contig and scaffold N50 values of 16.5 and 41.0 Mb, respectively. More than 98.2% of the 1,614 BUSCO genes were fully identified, and 25,939 genes were annotated, with 98.23% of them being functionally identifiable. Vigna angularis was estimated to diverge successively from Vigna unguiculata and Vigna radiata about 15.3 and 8.7 million years ago (Ma), respectively. This chromosome-level reference genome of Vigna angularis provides a robust foundation for exploring the functional genomics and genome evolution of adzuki bean, thereby facilitating advancements in molecular breeding of adzuki bean.

High accuracy meets high throughput for near full-length 16S ribosomal RNA amplicon sequencing on the Nanopore platform.

Small subunit (SSU) ribosomal RNA (rRNA) gene amplicon sequencing is a foundational method in microbial ecology. Currently, short-read platforms are commonly employed for high-throughput applications of SSU rRNA amplicon sequencing, but at the cost of poor taxonomic classification due to limited fragment lengths. The Oxford Nanopore Technologies (ONT) platform can sequence full-length SSU rRNA genes, but its lower raw-read accuracy has so-far limited accurate taxonomic classification and de novo feature generation. Here, we present a sequencing workflow, termed ssUMI, that combines unique molecular identifier (UMI)-based error correction with newer (R10.4+) ONT chemistry and sample barcoding to enable high throughput near full-length SSU rRNA (e.g. 16S rRNA) amplicon sequencing. The ssUMI workflow generated near full-length 16S rRNA consensus sequences with 99.99% mean accuracy using a minimum subread coverage of 3×, surpassing the accuracy of Illumina short reads. The consensus sequences generated with ssUMI were used to produce error-free de novo sequence features with no false positives with two microbial community standards. In contrast, Nanopore raw reads produced erroneous de novo sequence features, indicating that UMI-based error correction is currently necessary for high-accuracy microbial profiling with R10.4+ ONT sequencing chemistries. We showcase the cost-competitive scalability of the ssUMI workflow by sequencing 87 time-series wastewater samples and 27 human gut samples, obtaining quantitative ecological insights that were missed by short-read amplicon sequencing. ssUMI, therefore, enables accurate and low-cost full-length 16S rRNA amplicon sequencing on Nanopore, improving accessibility to high-resolution microbiome science.

Chromosome-level genome assembly of Tritrichomonas foetus, the causative agent of Bovine Trichomonosis

Tritrichomonas foetus is a parasitic protist responsible for bovine trichomonosis, a reproductive disease associated with significant economic burden to the livestock industry throughout the world. Here, we present a chromosome-level reference genome of T. foetus -KV-1 (ATCC 30924) using short-read (Illumina Miseq), long-read (Oxford Nanopore) and chromatin-linked (Hi-C) sequencing. This is the first chromosome-level genome of a parasitic protist of the order Tritrichomonadida and the second within the Parabasalia lineage, after Trichomonas vaginalis, the human-associated causative agent of the sexually transmitted infection in humans. Our constructed genome is 148 Mb in size, with a N50 length of the scaffolds of 22.9 Mb. The contigs are anchored in five super-scaffolds, corresponding to the expected five chromosomes of the species and covering 78% of the genome assembly. We predict 41,341 protein-coding genes, of which 95.10% have been functionally annotated. This high-quality genome assembly serves as a valuable reference genome for T. foetus to support future studies in functional genomics, genetic conservation and taxonomy.

A genome assembly and transcriptome atlas of the inbred Babraham pig to illuminate porcine immunogenetic variation.

The inbred Babraham pig serves as a valuable biomedical model for research due to its high level of homozygosity, including in the major histocompatibility complex (MHC) loci and likely other important immune-related gene complexes, which are generally highly diverse in outbred populations. As the ability to control for this diversity using inbred organisms is of great utility, we sought to improve this resource by generating a long-read whole genome assembly and transcriptome atlas of a Babraham pig. The genome was de novo assembled using PacBio longreads and error-corrected using Illumina shortreads. Assembled contigs were then mapped to the porcine reference assembly, Sscrofa11.1, to generate chromosome-level scaffolds. The resulting TPI_Babraham_pig_v1 assembly is nearly as contiguous as Sscrofa11.1 with a contig N50 of 34.95 Mb and contig L50 of 23. The remaining sequence gaps are generally the result of poor assembly across large and highly repetitive regions such as the centromeres and tandemly duplicated gene families, including immune-related gene complexes, that often vary in gene content between haplotypes. We also further confirm homozygosity across the Babraham MHC and characterize the allele content and tissue expression of several other immune-related gene complexes, including the antibody and T cell receptor loci, the natural killer complex, and the leukocyte receptor complex. The Babraham pig genome assembly provides an alternate highly contiguous porcine genome assembly as a resource for the livestock genomics community. The assembly will also aid biomedical and veterinary research that utilizes this animal model such as when controlling for genetic variation is critical.

PSLBII-27 Investigating the emergence of antimicrobial resistance in Mycoplasma bovis from feedlot cattle

Abstract Bovine respiratory disease (BRD) is the most significant disease affecting feedlot cattle in North America. Mycoplasma bovis is among the main BRD pathogenic bacteria. Infection control of M. bovis is hampered by a lack of effective vaccines and increasing antimicrobial resistance (AMR). Integrative and conjugative elements (ICEs) have been documented as a mechanism of horizontal gene transfer (HGT) in mycoplasmas. Mycoplasma ICEs (MICE) do not carry AMR genes but have been associated with the HGT of genes that confer AMR through point mutations. Hence, there is a need to gain a better understanding of the role of MICE in M. bovis AMR. Randomly selected M. bovis isolates (n = 54) collected from Western Canadian feedlot cattle (AB and SK) underwent whole-genome sequencing by short-reads (Illumina), with a subset (n = 6) selected based on AMR and ICE profiles for Oxford Nanopore long-read sequencing. Sequences were screened for the presence of MICE. MICE circularization (cMICE) is the first step of conjugation, and an in-house collection of 472 M. bovis field isolates (feedlot cattle just from AB) was screened by PCR for the presence of cMICE as a proxy for conjugation capabilities. Environmental conditions that could impact cMICE quantity, including growth phase, temperature, cell density, pH, starvation, UV light, CO2 concentration, and sub-inhibitory concentrations of mitomycin C were investigated by qPCR, followed by growth of M. bovis isolates under the most favorable conditions identified for cMICE-detection and screening by a high throughput qPCR method that eliminated the need for DNA extraction. Nineteen different mating pairs were carried out and 4 different environmental factors were tested for their possible impact on conjugation occurrence (incubation time, incubation volume, conjugation parent proportions, atmosphere). All isolates carried MICEs with 23/54 having a structure/sequence comparable to previously described functional MICE in mycoplasma, 43/54 possessing non-functional ICE, and 23 having >1 ICE. The presence of more than one MICE/genome suggests a lack of MICE (entry) exclusion systems in M. bovis, a trait that may promote the flow of genetic information among M. bovis isolates including AMR. Even though some factors including growth phase (early stationary phase) and pH (9.0) generated greater levels (≥1.5 fold) of cMICE, none of the examined parameters were statistically significant. However, as early stationary growth phase generated the greatest cMICE quantity (2.3 fold), M. bovis isolates were cMICE-screened at this growth stage for the expeditious identification of M. bovis conjugation candidates. A total of 25.7% M. bovis were cMICE-positive. Of 19 mating pairs tested, transconjugants were recovered from only 2 different mating pairs, possibly reflective of the need for further optimization of conjugation conditions.

COMPARISON OF COMPUTATIONAL METHODS FOR ANALYSING 16S rRNA SEQUENCING DATA PRODUCED WITH THE USE OF SHORT AND LONG READS

This research is driven by the importance of identifying species in microbial community for use in medical testing. Conventional methods including culturing, biochemical assays are often limited to the culturable organisms and do not provide information about relative abundance of different microbial taxa within a community. Comparative analysis of metagenomic programs method was used to identify the most appropriate option for long-read 16S rRNA data. Since the development of high-throughput sequencing technologies, novel methods of identifying species include taxonomic profiling of 16S rRNA. 16S rRNA gene contains variable regions (V1-V9) that are specific to different taxonomic groups. Illumina short-read sequencing technology provides read lengths 2*251 base pairs (bp) for paired-end sequencing, while fraction of 16S rRNA gene is around 1500 bp, thus reads can successfully cover 2 of 9 regions (300-400 bp), but not the whole gene. The Oxford Nanopore Technology (ONT) representing long-read sequencing or so-called 3rd generation sequencing is much more applicable for 16S rRNA profiling, as it can provide reads with length 1500 bp (exactly in line with the length of 16S rRNA gene). Extended read length provides high alignment quality making the downstream taxonomy classification and relative abundance calculation much easier and relevant. The samples from endometrium of uterus were studied in the first part of the research. Endometrial microbiome composition and balance can play an important role in the success of in vitro fertilization (IVF). Taxonomical classification was tested with NCBI, Silva databases. NCBI database consists of full genomes of Bacteria, Archaea and Eukaryote, Silva contains curated and annotated sequences of only small subunit ribosomal RNA of these domains. Among the enormous collection of metagenome classifiers there were selected three particular programs applying completely different algorithms: Emu, PathoScope and Kraken2. The comparative process was divided in three phases. In the first one, pipelines were run with three programs and 11 samples using Silva and NCBI databases, the results were compared both quantitively and visually. In the second phase, the results were compared to the short-read taxonomical classification, the discrepancies were analyzed and questions were put together for further investigation. During the third phase pipelines with best performed programs were run on sample with acknowledged microbial diversity and following metrics were assessed and visualized: True positive, False positive, Precision, Recall, F1-score, L1-norm, L2-norm. Results: After the first phase it was revealed that Kraken2, Emu, PathoScope produce very similar results, while MetaScope discrepancies exceeded substantially 10% threshold for 70% of the reads. Additionally, Kraken2 annotated significantly more genera upon the threshold than other programs indicating its low specificity. Consequently, these two programs were excluded from further analysis. The second phase brought to the conclusion that short-read sequencing reports reveal only one prevailing genus in the community, while long-read establish 3 of them, thus it was assumed that short-read classification is much less sensitive. In the third phase it was determined that Emu and PathoScope have least deviation from reference value of abundances (ground truth) and they successfully annotate all the species introduced in the sample. Therefore, the main outcome of the research is the recommendation to use Emu and PathoScope programs for the purpose of identifying species using long-read data.

Chromosome-level genome assembly of Oriental chestnut gall wasp (Dryocosmus kuriphilus)

Dryocosmus kuriphilus, commonly known as the chestnut gall wasp, belongs to the family Cynipidae and is native to China. It is a highly invasive insect species causing serious damage to chestnut trees and has rapidly spread to various continents, including Europe, North America, and Oceania. The D. kuriphilus has become one of the important pests of chestnut plants in the world and is listed as a quarantine object by the European and Mediterranean Plant Protection Organization (EPPO). In this study, we used PacBio long reads, Illumina short reads, and Hi-C sequencing data to construct a chromosome-level assembly of the D. kuriphilus genome. The assembled genome includes 14,729 contigs with a total length of 2.28 Gb and a contig N50 of 0.8 Mb. With Hi-C technology, 2.17 Gb (95.02%) of contigs were anchored and oriented into the 10 pseudochromosomes with the scaffold N50 of 198.8 Mb and the scaffold N90 of 158.8 Mb. In total, 24,086 protein-coding genes were predicted in the assembled D. kuriphilus genome as the reference gene set. A total of 1.82 Gb repeats (occupying 79.7% of the genome), including 1.42 Gb of transposable elements and 0.40 Gb of tandem repeats, were identified in D. kuriphilus genome. In the evaluation of completeness, the BUSCO analysis determined a level of 98.1% completeness for the assembled genome sequences based on the Insecta database (OrthoDB version 10). The high-quality genome assembly of D. kuriphilus will not only provide a valuable reference for the study of its evolutionary history and genetic structure but also facilitate the research of host-pest interactions and invasiveness. Moreover, this genome assembly will promote in the development of effective management strategies to mitigate the economic and ecological impacts of this invasive pest on chestnut trees and ecosystems.

Infectious bronchitis virus vaccination, but not the presence of XCR1, is correlated with large differences in chicken caecal microbiota.

The chicken immune system and microbiota play vital roles in maintaining gut homeostasis and protecting against pathogens. In mammals, XCR1+ conventional dendritic cells (cDCs) are located in the gut-draining lymph nodes and play a major role in gut homeostasis. These cDCs sample antigens in the gut luminal contents and limit the inflammatory response to gut commensal microbes by generating appropriate regulatory and effector T-cell responses. We hypothesized that these cells play similar roles in sustaining gut homeostasis in chickens, and that chickens lacking XCR1 were likely to contain a dysbiotic caecal microbiota. Here we compare the caecal microbiota of chickens that were either heterozygous or homozygous XCR1 knockouts, that had or had not been vaccinated for infectious bronchitis virus (IBV). We used short-read (Illumina) and long-read (PacBio HiFi) metagenomic sequencing to reconstruct 670 high-quality, strain-level metagenome assembled genomes. We found no significant differences between alpha diversity or the abundance of specific microbial taxa between genotypes. However, IBV vaccination was found to correlate with significant differences in the richness and beta diversity of the microbiota, and to the abundance of 40 bacterial genera. In conclusion, we found that a lack of XCR1 was not correlated with significant changes in the chicken microbiota, but IBV vaccination was.

Annotation and Characterization of the Zacco platypus Genome

The pale chub Zacco platypus (Cypriniformes; Xenocyprididae; Jordan & Evermann, 1902) is widely distributed across freshwater ecosystems in East Asia and has been recognized as a potential model fish species for ecotoxicology and environmental monitoring. Here, a high-quality de novo genome assembly of Z. platypus was constructed through the integration of a combination of long-read Pacific Bioscience (PacBio) sequencing, short-read Illumina sequencing, and Hi-C sequencing technologies. Z. platypus has the smallest genome size compared to other species belonging to the order Cypriniformes. The assembled genome encompasses 41.45% repeat sequences. As shown in other fish, a positive correlation was observed between genome size and the composition of transposable elements (TE) in the genome. Among TEs, a relatively higher rate of DNA transposon was observed, which is a common pattern in the members of the order Cypriniformes. Functional annotation was processed using four representative databases, identifying a core set of 12,907 genes shared among them. Orthologous gene family analysis revealed that Z. platypus has experienced more gene family contraction rather than expansion compared to other Cypriniformes species. Among the uniquely expanded gene families in Z. platypus, detoxification and stress-related gene families were identified, suggesting that this species could represent a promising model for ecotoxicology and environmental monitoring. Taken together, the Z. platypus genome assembly will provide valuable data for omics-based health assessments in aquatic ecosystems, offering further insights into the environmental and ecological facets within this species.

Genome sequencing and assembly of Indian major carp, Cirrhinus mrigala (Hamilton, 1822)

The freshwater aquaculture species, Cirrhinus mrigala, commonly known as mrigal, holds significance in the carp-dominated aquaculture system, globally. Despite constituting 1.08% of the total freshwater aquaculture production, mrigal is the third most important Indian major carp. However, its genome and associated information is not available. This study aims to address this gap by generation high quality genome assembly using PacBio long reads, Illumina short reads and Hi-C scaffolding. The characterization of assembled highly contiguous genome, 1.057 Gb in size, revealed 39,091 genes with functional annotations. The orthology analysis based on direct orthologs and single copy ortholgs places C. mrigala in a distinct position within the Otophysi clade. Additionally, the study delves into Hox gene clusters, identifying 38 Hox genes distributed in seven clusters. The present genomic information offers potential applications for sustainable aquaculture management, including selection programs for economic traits.

Chromosome-level genome assembly of the invasive pest Pseudococcus jackbeardsleyi (Hemiptera: Pseudococcidae)

Among over 2,000 species of mealybugs (Hemiptera: Pseudococcidae), only 13 genomes have been published so far, seriously limiting the researches on the phylogeny and adaptive evolution of this group. The continuous publication of mealybug genomes will significantly facilitate our exploration of the biological characteristics, detrimental attributes, and control strategies of the Pseudococcidae family. Jack Beardsley mealybug (Pseudococcus jackbeardsleyi) as one of the hazardous invasive pests, it could cause enormous losses to the fruit and vegetable industries worldwide. Herein, we combined Nanopore long-read, short-read Illumina and Hi-C sequencing, generating a high-quality chromosome-level genome assembly of P. jackbeardsleyi. The genome size was determined to be 334.818 Mb, which was assembled into 5 linkage groups with a N50 of 67.233 Mb. The BUSCO analysis demonstrated the completeness of the genome assembly and annotation are 95.7% and 92.8%, respectively. The developed high-quality genome will serve as an asset for delving into the genetic mechanisms underlying the invasiveness of P. jackbeardsleyi, thereby offering a crucial theoretical foundation for the prevention and management of Pseudococcidae pests.

A chromosomal-level genome assembly of Serrognathus titanus Boisduval, 1835 (Coleoptera: Lucanidae)

The Stag beetle (Coleoptera: Lucanidae) is a fascinating group, often considered one of the most primitive within the Scarabaeoidea. They are valuable models for studying beetle evolution. However, the lack of high-quality genomes hinders our understanding of the evolution and ecology of Lucanidae. In this study, we present a chromosome-level genome of Serrognathus titanus by combining PacBio HiFi long reads, Illumina short reads, and Hi-C data. The genome spans 384.07 Mb, with a scaffold N50 size of 75.81 Mb, and most contigs (97.45%, 374.30 Mb) were anchored into six chromosomes. Our BUSCO analysis of the assembly indicates a completeness of 97.6% (n = 1,367), with 92.8% single-copy BUSCOs and 4.8% duplicated BUSCOs identified. Additionally, we found that the genome contains 43.87% (168.50 Mb) repeat elements and identified 14,263 predicted protein-coding genes. The high-quality genome of S. titanus provides valuable genomic information for comprehending the evolution and ecology of Lucanidae.

A hybrid genome assembly of the endangered aye-aye (Daubentonia madagascariensis).

The aye-aye (Daubentonia madagascariensis) is the only extant member of the Daubentoniidae primate family. Although several reference genomes exist for this endangered strepsirrhine primate, the predominant usage of short-read sequencing has resulted in limited assembly contiguity and completeness, and no protein-coding gene annotations have yet been released. Here, we present a novel, fully annotated, chromosome-level hybrid de novo assembly for the species based on a combination of Oxford Nanopore Technologies long reads and Illumina short reads and scaffolded using genome-wide chromatin interaction data-a community resource that will improve future conservation efforts as well as primate comparative analyses.

Escherichia coli novel sequence type 11873 harbours a new CTX-M-15–carrying multidrug resistance type 1/2 hybrid IncC plasmid

ObjectiveThe aim of the current study was to determine the genomic map of the resistance genes of two CTX-M-15–carrying Escherichia coli strains belonging to novel sequence type (ST) 11873. Complete, closed genome sequences of the E. coli strains were obtained by applying a combination of short-read Illumina and long-read Oxford Nanopore-based sequencing. MethodsIsolation of E. coli was performed using ECC CHROMagar and antibiotic sensitivity patterns were determined using Sensititre EUVSEC plates. Whole-genome sequencing was performed for two E. coli strains (3–338 and 5–325) using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. ResultsThe complete genome of strain 3–338 (GenBank accession no. CP130007–17) was assembled into a circular chromosome of 4.65 Mb and 10 plasmids (between 2 and 148 kb). Strain 5–325 (CP130018–27) exhibited a circular chromosome of 4.7 Mb and 9 plasmids (between 2 and 148 kb). Both strains carried an identical type 1/2 hybrid IncC plasmid (∼148 kb) harbouring multiple antibiotic resistance genes (ARGs), including blaCTX−M-15, blaOXA-1, blaTEM-1, qnrS1, sul2, aphA1, aacC2, mph(A) and floR. This plasmid also carried heavy metal resistance genes, such as chrA and arsR. Strain 5–325 carried an additional IncFIB plasmid (∼78 kb) harbouring additional ARGs, including blaTEM-1, qnrS1, tet(A), dfrA14, sul2, strA and strB. ConclusionsOur study shows the emergence of a CTX-M-15–carrying type 1/2 hybrid IncC plasmid in novel E. coli ST11873. These findings emphasise the need for population-based sewage surveillance for understanding the prevalence of antibiotic resistance in pathogens in order to mitigate the further spread of such resistance factors.

A multiomic characterization of the leukemia cell line REH using short- and long-read sequencing.

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1 Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.