Long-read Whole-genome Sequencing Research Articles

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow.MethodsWe developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case–control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES).ResultsUsing our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES.ConclusionsOur ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

Abstract 6247: Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) and linitis-plastica type gastric cancer (STAD) are the most representative refractory cancers even in the era of molecularly targeted therapy and immunotherapy. Extensive characterization of these tumors, especially regarding the genomic and epigenomic characteristics of pure cancer cells, has been hampered by the high stromal content in the primary tumor samples. Here we took advantage of cancer cells purified from malignant ascites and established corresponding cell lines to overcome the limitations associated with the analysis and to discover their vulnerabilities. Methods: We developed cell lines from malignant ascitic fluid samples obtained from 60 patients with PDAC and 59 patients with linitis-plastica type STAD. We performed comprehensive multi-omic analyses of these cell lines using long-read whole-genome sequencing (WGS) for complex somatic genomic alterations and for methylation aberration. We also conducted long-read RNA sequencing (iso-seq) for full-length isoform exploration, and chromatin immunoprecipitation coupled with sequencing (ChIP-seq) for enhancer analysis. Complementary short-read whole-genome sequencing and RNA sequencing were also performed. Results: Comprehensive transcriptome analyses identified a cluster with high epithelial-mesenchymal transition (EMT) characteristics, consisting of the majority of the PDAC cell lines and a half of the STAD cell lines. ChIP-seq analysis against H3K27ac revealed the distinct transcriptional circuit that generated EMT cluster-specific super-enhancers governed by SMAD3, independent of cancer type. Most of the transcriptional activity was controlled by the gene methylation. We also identified the differential expression levels of the splicing factors between the EMT-activated and the non-activated groups. Comprehensive isoform analysis by iso-seq revealed that the isoform usage was completely different between the EMT group and the other, especially in genes of the actomyosin pathway which confer characteristic features of the EMT cluster in the cell morphology and cell motility. Using the CHM13 reference genome, long-read WGS analyses revealed the recurrent structural alterations specific to the EMT group such as those involving CDKN2A. We also identified PDAC-specific structural alterations. Conclusion: We identified the distinct molecular characteristics of the EMT-activated group in PDAC and linitis-plastica type STAD which may confer the therapeutic resistance. Our findings provide an interesting insight for the further innovation of the treatment strategy for these intractable cancers. Citation Format: Yosuke Tanaka, Hiroyuki Mano. Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6247.

Abstract 1751: Deep multi-omics analysis of uterine leiomyosarcoma reveals defective HRD signature, a novel gene fusion and the amplification of the poor-prognosis CTHRC1 gene

Abstract Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer representing approximately 2-5% of all uterine malignancies. The molecular heterogeneity and pathogenesis of uLMS are notwell understood. To unravel the intricate molecular landscape of uLMS, we conducted a multi-omics workflow consisting of 4 high-throughput sequencing technologies; short- and long-readwhole genome sequencing (WGS), RNA-Seq, and Tandem mass tag (TMT) labeling enablingmassively parallel proteome proteomic analyses. Our study cohort consisted of 97 fresh frozentissue samples collected from 68 female patients, with 19 uLMS, 37 leiomyoma (LM), and 41normal myometrium (MM). A deep analysis of somatic variants with clinical impact revealed 46variants present in 90% of samples and two actionable therapeutic targets: IDH1_p.Arg132Cysand KRAS_p.Gly12Val. Also, 80% of the samples presented a chromothripsis signature and 60%presented the SBS3 COSMIC signature related to homologous recombination deficiency (HRD).Analysis of the proliferation score in RNA-Seq data revealed a notable difference among thetumor groups, signifying a higher proliferation index in the uLMS group, suggesting a moreaggressive growth pattern with implications for clinical behavior and disease progression. Afterapplying a customized workflow to identify balanced chromosomal abnormalities of genefusions with clinical impact, the proto-oncogene eukaryotic elongation factor 1 alpha (EEF1A1)emerged as a prominent fusion partner. Eight fusions in 7 samples were identified withtranscriptional overexpression in positive cases. At the proteomic level the enrichment analysisunveiled activated pathways associated with the innate immune system, while suppressedpathways pertained to extracellular matrix (ECM) organization and smooth muscle contraction.With an integrated bioinformatics pipeline, we identified copy number amplification of theCTHRC1 gene in 80% of the samples, accompanied by increased gene expression and proteinlevels. This gene is related to collagen remodeling and degradation and has been demonstratedto be upregulated in some solid tumors and is closely associated with malignancy. Survivalcurve analysis highlighted a significantly poorer prognosis in uLMS exhibiting CTHRC1 geneamplification with potential clinical relevance of CTHRC1 amplification as a prognostic indicatorin uLMS. Taken together these results show the overall suppression of ECM and collagenremodeling in uLMS associated with poor prognosis. Additionally, these data provide the firstevidence that the EEF1A1 gene has a strong selective pressure in uLMS underscoring theindispensable role of precision genomics in advancing precision medicine. Citation Format: Raul Maia Falcao, Jorge Estefano de Souza, William Mathieson, Joseph Woodward Carlson, Tirzah Braz Petta. Deep multi-omics analysis of uterine leiomyosarcoma reveals defective HRD signature, a novel gene fusion and the amplification of the poor-prognosis CTHRC1 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1751.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Plasmid-encoded gene duplications of extended-spectrum β-lactamases in clinical bacterial isolates.

The emergence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.

Long-read sequencing reveals the structural complexity of genomic integration of HPV DNA in cervical cancer cell lines

BackgroundCervical cancer (CC) causes more than 311,000 deaths annually worldwide. The integration of human papillomavirus (HPV) is a crucial genetic event that contributes to cervical carcinogenesis. Despite HPV DNA integration is known to disrupt the genomic architecture of both the host and viral genomes in CC, the complexity of this process remains largely unexplored.ResultsIn this study, we conducted whole-genome sequencing (WGS) at 55-65X coverage utilizing the PacBio long-read sequencing platform in SiHa and HeLa cells, followed by comprehensive analyses of the sequence data to elucidate the complexity of HPV integration. Firstly, our results demonstrated that PacBio long-read sequencing effectively identifies HPV integration breakpoints with comparable accuracy to targeted-capture Next-generation sequencing (NGS) methods. Secondly, we constructed detailed models of complex integrated genome structures that included both the HPV genome and nearby regions of the human genome by utilizing PacBio long-read WGS. Thirdly, our sequencing results revealed the occurrence of a wide variety of genome-wide structural variations (SVs) in SiHa and HeLa cells. Additionally, our analysis further revealed a potential correlation between changes in gene expression levels and SVs on chromosome 13 in the genome of SiHa cells.ConclusionsUsing PacBio long-read sequencing, we have successfully constructed complex models illustrating HPV integrated genome structures in SiHa and HeLa cells. This accomplishment serves as a compelling demonstration of the valuable capabilities of long-read sequencing in detecting and characterizing HPV genomic integration structures within human cells. Furthermore, these findings offer critical insights into the complex process of HPV16 and HPV18 integration and their potential contribution to the development of cervical cancer.

HAT: de novo variant calling for highly accurate short-read and long-read sequencing data.

de novo variants (DNVs) are variants that are present in offspring but not in their parents. DNVs are both important for examining mutation rates as well as in the identification of disease-related variation. While efforts have been made to call DNVs, calling of DNVs is still challenging from parent-child sequenced trio data. We developed Hare And Tortoise (HAT) as an automated DNV detection workflow for highly accurate short-read and long-read sequencing data. Reliable detection of DNVs is important for human genomics and HAT addresses this need. HAT is a computational workflow that begins with aligned read data (i.e. CRAM or BAM) from a parent-child sequenced trio and outputs DNVs. HAT detects high-quality DNVs from Illumina short-read whole-exome sequencing, Illumina short-read whole-genome sequencing, and highly accurate PacBio HiFi long-read whole-genome sequencing data. The quality of these DNVs is high based on a series of quality metrics including number of DNVs per individual, percent of DNVs at CpG sites, and percent of DNVs phased to the paternal chromosome of origin. https://github.com/TNTurnerLab/HAT.

Prolonged carriage of OXA-244-carbapenemase-producing Escherichia coli complicates epidemiological investigations

The rapid increase of OXA-244-producing Escherichia coli, predominantly driven by genetically clustered isolates of sequence type (ST)38, has been observed in at least nine European countries, including Germany. However, the reasons for the spread of OXA-244-producing E. coli remain unclear. Here, we aim to evaluate the possibility of prolonged carriage. We identified a total of six different patients with repeated detection of OXA-244-producing E. coli isolates, which were subjected to both short and long-read whole-genome sequencing (WGS). Besides allelic differences using core genome multilocus sequence typing (cgMLST) analyses, we obtained numbers of single-nucleotide polymorphisms (SNPs) to calculate individual base-pair substitution (BPS) rates. To assess possible re-exposure and risk factors for prolonged carriage, case interviews were conducted. The time between detections ranged from eleven months to more than three years. Initial isolates originated in three+ out of six cases from clinical samples, whereas remaining samples were from screening, mostly in the inpatient setting. As expected, cgMLST analyses showed low numbers of allelic differences between isolates of each case ranging from 1 to 4, whereas numbers of SNPs were between 2 and 99 (mean = 36), thus clearly highlighting the discrepancy between these different bacterial typing approaches. For five out of six cases, observed BPS rates suggest that patients can be colonized with OXA-244-producing E. coli, including ST38 cluster isolates, for extensively long times. Thus, we may have previously missed the epidemiological link between cases because exposure to OXA-244-producing E. coli could have occurred in a time frame, which has not been evaluated in previous investigations. Our results may help to guide future epidemiological investigations as well as to support the interpretation of genetic diversity of OXA-244-producing E. coli, particularly among ST38 cluster isolates.

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.

Genotypic characterization of Streptococcus didelphis causative of fatal infection in white-eared opossums.

Streptococcus didelphis was reported once as related to severe infections in opossums. Thus, we present the first comprehensive whole-genome characterization of clinical S. didelphis strains isolated from white-eared opossums (Didelphis albiventris). Long-read whole-genome sequencing was performed using the MinION platform, which allowed the prediction of several genomic features. We observed that S. didelphis genomes harbor a cluster for streptolysin biosynthesis, and a conserved genomic island with genes involved in transcriptional regulation (arlR) and transmembrane transport (bcrA). Antimicrobial resistance genes for several drug classes were found including beta-lactam, which is the main antimicrobial class used in Streptococcus spp. infections; however no phenotypical resistance was observed. In addition, we predicted the presence of 33 virulence factors in the analyzed genomes. High phylogenetic similarity was observed between clinical and reference strains, yet no clonality was suggested. We also proposed dnaN, gki, pros, and xpt as housekeeping candidates to be used in S. didelphis sequence typing. This is the first whole-genome characterization of S. didelphis, whose data provides important insights on its pathogenicity.

A large pedigree study confirmed the CGG repeat expansion of RILPL1 Is associated with oculopharyngodistal myopathy

BackgroundOculopharyngodistal myopathy (OPDM) is an autosomal dominant adult-onset degenerative muscle disorder characterized by ptosis, ophthalmoplegia and weakness of the facial, pharyngeal and limb muscles. Trinucleotide repeat expansions in non-coding regions of LRP12, G1PC1, NOTCH2NLC and RILPL1 were reported to be the etiologies for OPDM.ResultsIn this study, we performed long-read whole-genome sequencing in a large five-generation family of 156 individuals, including 21 patients diagnosed with typical OPDM. We identified CGG repeat expansions in 5’UTR of RILPL1 gene in all patients we tested while no CGG expansion in unaffected family members. Repeat-primed PCR and fluorescence amplicon length analysis PCR were further confirmed the segregation of CGG expansions in other family members and 1000 normal Chinese controls. Methylation analysis indicated that methylation levels of the RILPL1 gene were unaltered in OPDM patients, which was consistent with previous studies. Our findings provide evidence that RILPL1 is associated OPDM in this large pedigree.ConclusionsOur results identified RILPL1 is the associated the disease in this large pedigree.

Copy number variations and their effect on the plasma proteome.

Structural variations, including copy number variations (CNVs), affect around 20 million bases in the human genome and are common causes of rare conditions. CNVs are rarely investigated in complex disease research because most CNVs are not targeted on the genotyping arrays or the reference panels for genetic imputation. In this study, we characterize CNVs in a Swedish cohort (N = 1,021) using short-read whole-genome sequencing (WGS) and use long-read WGS for validation in a subcohort (N = 15), and explore their effect on 438 plasma proteins. We detected 184,182 polymorphic CNVs and identified 15 CNVs to be associated with 16 proteins (P < 8.22×10-10). Of these, 5 CNVs could be perfectly validated using long-read sequencing, including a CNV which was associated with measurements of the osteoclast-associated immunoglobulin-like receptor (OSCAR) and located upstream of OSCAR, a gene important for bone health. Two other CNVs were identified to be clusters of many short repetitive elements and another represented a complex rearrangement including an inversion. Our findings provide insights into the structure of common CNVs and their effects on the plasma proteome, and highlights the importance of investigating common CNVs, also in relation to complex diseases.

Identification of genetic risk loci and causal insights associated with Parkinson's disease in African and African admixed populations: a genome-wide association study

An understanding of the genetic mechanisms underlying diseases in ancestrally diverse populations is an important step towards development of targeted treatments. Research in African and African admixed populations can enable mapping of complex traits, because of their genetic diversity, extensive population substructure, and distinct linkage disequilibrium patterns. We aimed to do a comprehensive genome-wide assessment in African and African admixed individuals to better understand the genetic architecture of Parkinson's disease in these underserved populations. We performed a genome-wide association study (GWAS) in people of African and African admixed ancestry with and without Parkinson's disease. Individuals were included from several cohorts that were available as a part of the Global Parkinson's Genetics Program, the International Parkinson's Disease Genomics Consortium Africa, and 23andMe. A diagnosis of Parkinson's disease was confirmed clinically by a movement disorder specialist for every individual in each cohort, except for 23andMe, in which it was self-reported based on clinical diagnosis. We characterised ancestry-specific risk, differential haplotype structure and admixture, coding and structural genetic variation, and enzymatic activity. We included 197 918 individuals (1488 cases and 196 430 controls) in our genome-wide analysis. We identified a novel common risk factor for Parkinson's disease (overall meta-analysis odds ratio for risk of Parkinson's disease 1·58 [95% CI 1·37-1·80], p=2·397 × 10-14) and age at onset at the GBA1 locus, rs3115534-G (age at onset β=-2·00 [SE=0·57], p=0·0005, for African ancestry; and β=-4·15 [0·58], p=0·015, for African admixed ancestry), which was rare in non-African or non-African admixed populations. Downstream short-read and long-read whole-genome sequencing analyses did not reveal any coding or structural variant underlying the GWAS signal. The identified signal seems to be associated with decreased glucocerebrosidase activity. Our study identified a novel genetic risk factor in GBA1 in people of African ancestry, which has not been seen in European populations, and it could be a major mechanistic basis of Parkinson's disease in African populations. This population-specific variant exerts substantial risk on Parkinson's disease as compared with common variation identified through GWAS and it was found to be present in 39% of the cases assessed in this study. This finding highlights the importance of understanding ancestry-specific genetic risk in complex diseases, a particularly crucial point as the Parkinson's disease field moves towards targeted treatments in clinical trials. The distinctive genetics of African populations highlights the need for equitable inclusion of ancestrally diverse groups in future trials, which will be a valuable step towards gaining insights into novel genetic determinants underlying the causes of Parkinson's disease. This finding opens new avenues towards RNA-based and other therapeutic strategies aimed at reducing lifetime risk of Parkinson's disease. The Global Parkinson's Genetics Program, which is funded by the Aligning Science Across Parkinson's initiative, and The Michael J Fox Foundation for Parkinson's Research.

Identification of small-sized intrachromosomal segments at the ends of INV-DUP-DEL patterns.

The mechanism of chromosomal rearrangement associated with inverted-duplication-deletion (INV-DUP-DEL) pattern formation has been investigated by many researchers, and several possible mechanisms have been proposed. Currently, fold-back and subsequent dicentric chromosome formation has been established as non-recurrent INV-DUP-DEL pattern formation mechanisms. In the present study, we analyzed the breakpoint junctions of INV-DUP-DEL patterns in five patients using long-read whole-genome sequencing and detected 2.2-6.1 kb copy-neutral regions in all five patients. At the end of the INV-DUP-DEL, two patients exhibited chromosomal translocations, which are recognized as telomere capture, and one patient showed direct telomere healing. The remaining two patients had additional small-sized intrachromosomal segments at the end of the derivative chromosomes. These findings have not been previously reported but they may only be explained by the presence of telomere capture breakage. Further investigations are required to better understand the mechanisms underlying this finding.

Elucidating causative gene variants in hereditary Parkinson’s disease in the Global Parkinson’s Genetics Program (GP2)

The Monogenic Network of the Global Parkinson’s Genetics Program (GP2) aims to create an efficient infrastructure to accelerate the identification of novel genetic causes of Parkinson’s disease (PD) and to improve our understanding of already identified genetic causes, such as reduced penetrance and variable clinical expressivity of known disease-causing variants. We aim to perform short- and long-read whole-genome sequencing for up to 10,000 patients with parkinsonism. Important features of this project are global involvement and focusing on historically underrepresented populations.

Mobilization of a novel diatom Mutator-Like Element (MULE) transposon inactivates the uridine monophosphate synthase (UMPS) locus in Phaeodactylum tricornutum.

Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often result in peculiar biochemical and biological traits. Transposable elements (TE) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and centrally contribute to genome evolution. Here, through long-read whole genome sequencing, we identified a novel Mutator-Like Element (MULE) in the model diatom Phaeodactylum tricornutum, and we report the direct observation of its mobilization within the course of a single laboratory experiment. Under selective conditions, this novel TE inactivated the uridine monophosphate synthase (UMPS) gene of P. tricornutum, one of the few endogenous genetic loci currently targeted for selectable auxotrophy for functional genetics and genome editing applications. We report the observation of a recently mobilized transposon in diatoms with unique features. These include the combined presence of a MULE transposase with Zinc finger, SWIM-type domains, and of a diatom-specific E3 ubiquitin ligase of the zinc finger UBR type, which indicate a novel mobilization mechanism. Our findings provide new elements for the understanding of the role of TEs in diatom genome evolution and in the enrichment of intraspecific genetic variability.

New Delhi metallo-β-lactamase–producing Escherichia coli among dogs at an animal rescue facility—Wisconsin, 2022

Background: New Delhi Metallo-β-lactamase (NDM)–producing Escherichia coli are highly resistant organisms that spread quickly. In the United States, organisms with blaNDM are rare and mostly associated with healthcare settings. However, in other countries, blaNDM can be relatively common and are found in community settings. State veterinary and public health partners detected NDM E. coli in a dog from Iran living at a Wisconsin animal rescue facility (ARF), where 40% of dogs had international origins. We investigated to determine spread among dog and human contacts and prevent further transmission. Methods: We screened dogs and humans at the ARF, a local veterinary clinic (clinic A), and ARF staff homes (homes A and B) for colonization with blaNDM. We reviewed veterinary records and conducted a case–control analysis to identify risk factors for blaNDM acquisition among dogs. We evaluated ARF infection control practices. Screening specimens that were positive for blaNDM were cultured. We conducted an analysis of short- and long-read whole-genome sequencing data to evaluate isolate relatedness. We compared NDM E. coli sequences from dogs to all NDM E. coli sequences from humans collected in Wisconsin and nearby states. Results: Screening identified blaNDM colonization in 27 (37%) of 73 ARF dogs and 4 (56%) of 7 dogs in home A, but not in ARF or staff in clinic A. Among ARF dogs with blaNDM, 20 (74%) 27 had international origins and 22 (81%) had ≥1 medical condition. Dogs sharing the same space (OR, 5.1; 95% CI, 1.8–14.7) were associated with blaNDM acquisition. We observed high animal density, soiled environments, and insufficient hand hygiene. ARF staff wore workwear and work shoes off site, including to home A. Sequencing identified 3 multilocus sequence types (STs) using the Achtman scheme among 27 isolates with blaNDM-5. Most isolates were ST361 (20 of 27, 74%) followed by ST167 (6 of 27, 22%) and ST1163 (1 of 27, 4%). Within-MLST cluster variability was &lt;1–3 high-quality single-nucleotide variant differences, each harboring a ST-specific plasmid with blaNDM-5. No NDM-E. coli sequences from humans appeared related. Conclusions: Investigation of a single isolate led to identification of widespread NDM-E. coli transmission among dogs at an ARF. There were multiple NDM E. coli introductions to the ARF, likely by dogs of international origin. Poor hygiene contributed to transmission among ARF dogs and to dogs outside the ARF. Transmission of blaNDM-5 at the ARF and offsite spread to home A demonstrate the potential for unrecognized community sources to disseminate NDM E. coli in community settings. Strategies and lessons learned from interventions to prevent antibiotic resistance in human healthcare settings may inform and support prevention in animal care.Disclosures: None

Efflux-Related Carbapenem Resistance in Acinetobacter baumannii Is Associated with Two-Component Regulatory Efflux Systems' Alteration and Insertion of ΔAbaR25-Type Island Fragment.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

Exchange of Carbapenem-Resistant Escherichia coli Sequence Type 38 Intercontinentally and among Wild Bird, Human, and Environmental Niches.

Carbapenem-resistant Enterobacteriaceae (CRE) are a global threat to human health and are increasingly being isolated from nonclinical settings. OXA-48-producing Escherichia coli sequence type 38 (ST38) is the most frequently reported CRE type in wild birds and has been detected in gulls or storks in North America, Europe, Asia, and Africa. The epidemiology and evolution of CRE in wildlife and human niches, however, remains unclear. We compared wild bird origin E. coli ST38 genome sequences generated by our research group and publicly available genomic data derived from other hosts and environments to (i) understand the frequency of intercontinental dispersal of E. coli ST38 clones isolated from wild birds, (ii) more thoroughly measure the genomic relatedness of carbapenem-resistant isolates from gulls sampled in Turkey and Alaska, USA, using long-read whole-genome sequencing and assess the spatial dissemination of this clone among different hosts, and (iii) determine whether ST38 isolates from humans, environmental water, and wild birds have different core or accessory genomes (e.g., antimicrobial resistance genes, virulence genes, plasmids) which might elucidate bacterial or gene exchange among niches. Our results suggest that E. coli ST38 strains, including those resistant to carbapenems, are exchanged between humans and wild birds, rather than separately maintained populations within each niche. Furthermore, despite close genetic similarity among OXA-48-producing E. coli ST38 clones from gulls in Alaska and Turkey, intercontinental dispersal of ST38 clones among wild birds is uncommon. Interventions to mitigate the dissemination of antimicrobial resistance throughout the environment (e.g., as exemplified by the acquisition of carbapenem resistance by birds) may be warranted. IMPORTANCE Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene blaOXA-48. This is the most frequently reported carbapenem-resistant clone in wild birds, though it was unclear if it circulated within wild bird populations or was exchanged among other niches. The results from this study suggest that E. coli ST38 strains, including those resistant to carbapenems, are frequently exchanged among wild birds, humans, and the environment. Carbapenem-resistant E. coli ST38 clones in wild birds are likely acquired from the local environment and do not constitute an independent dissemination pathway within wild bird populations. Management actions aimed at preventing the environmental dissemination and acquisition of antimicrobial resistance by wild birds may be warranted.