Long-read Whole-genome Sequencing Research Articles

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 <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.

Rapid detection of mutations and chromosomal rearrangements in T-DNA insertion mutants of Colletotrichum higginsianum using long-read whole-genome sequencing

Rapid detection of mutations and chromosomal rearrangements in T-DNA insertion mutants of Colletotrichum higginsianum using long-read whole-genome sequencing

CAG Repeat Expansion in THAP11 Is Associated with a Novel Spinocerebellar Ataxia.

More than 50 loci are associated with spinocerebellar ataxia (SCA), and the most frequent subtypes share nucleotide repeats expansion, especially CAG expansion. The objective of this study was to confirm a novel SCA subtype caused by CAG expansion. We performed long-read whole-genome sequencing combined with linkage analysis in a five-generation Chinese family, and the finding was validated in another pedigree. The three-dimensional structure and function of THAP11 mutant protein were predicted. Polyglutamine (polyQ) toxicity of THAP11 gene with CAG expansion was assessed in skin fibroblasts of patients, human embryonic kidney 293 and Neuro-2a cells. We identified THAP11 as the novel causative SCA gene with CAG repeats ranging from 45 to 100 in patients with ataxia and from 20 to 38 in healthy control subjects. Among the patients, the number of CAA interruptions within CAG repeats was decreased to 3 (up to 5-6 in controls), whereas the number of 3' pure CAG repeats was up to 32 to 87 (4-16 in controls), suggesting that the toxicity of polyQ protein was length dependent on the pure CAG repeats. Intracellular aggregates were observed in cultured skin fibroblasts from patients. THAP11 polyQ protein was more intensely distributed in the cytoplasm of cultured skin fibroblasts from patients, which was replicated with in vitro cultured neuro-2a transfected with 54 or 100 CAG repeats. This study identified a novel SCA subtype caused by intragenic CAG repeat expansion in THAP11 with intracellular aggregation of THAP11 polyQ protein. Our findings extended the spectrum of polyQ diseases and offered a new perspective in understanding polyQ-mediated toxic aggregation. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

Abstract 4327: MSI cancer associated DNA (TA)n-dinucleotide repeat expansions and implications for Werner synthetic lethality

Abstract Microsatellite instability (MSI) is caused by deficient DNA mismatch repair (MMR) and is a ubiquitous feature of cancer. Werner syndrome (WRN) helicase is involved in genome stability and DNA repair. We identified WRN as a synthetic-lethal target in dMMR/MSI cancers and highlighted WRN inhibition as a therapeutic option for dMMR/MSI cancers refractory to available therapies. A previously unappreciated genetic feature of dMMR/MSI cancer cells, DNA (TA)n-dinucleotide repeat expansions, were recently reported to cause vulnerability to WRN depletion. Our mechanistic understanding of TA-dinucleotide repeat expansion biology is limited, and their potential therapeutic implications are unclear. To investigate the landscape of these alterations in cancer, we inferred (TA)-dinucleotide repeat expansions by performing coverage analysis in a collection of hundreds of preclinical cancer models and human tumors (PCAWG) profiled by whole genome sequencing (WGS). We validated our findings in cancer cell lines and organoid cultures by performing long-read WGS. Furthermore, we investigated TA-expansions in single-cell-derived clones from human MSI tumors and cancer organoids. Finally, we inferred TA repeats in laser capture microdissection (LCMB)-derived samples obtained from patients affected by familial cancer predisposition syndromes. Our analysis unveils the landscape of TA-repeats alterations in a large collection of tumors and preclinical models, informing on the level of inter-tumor heterogeneity and their association with variable levels of WRN dependency. In addition, we investigated intra-patient tumor heterogeneity of TA-repeats length both within clonal organoids expanded from normal and neoplastic colorectal stem cells, and within different subclones derived from MSI cancer organoids. Furthermore, analysis of non-neoplastic and neoplastic tissues from patients affected by familial cancer predisposition syndromes revealed the pattern of TA-repeats expansions associated with various DNA-repair pathway alterations. Finally, we will discuss the clinical implications of our findings, as TA-repeats heterogeneity may affect sensitivity and resistance to the future generation of WRN inhibitors. Our data provide fresh insights into the inter and intra-tumoral heterogeneity of TA-dinucleotide repeat expansions in human cancers. These data contribute to understanding the role of MMR in cancer and exploiting Werner as a therapeutic target in cancer. Citation Format: Gabriele Picco, Shriram Bhosle, Maria Kalyva, Elena Grassi, Freddy Gibson, Angham Al Saedi, Sara Vieira, Mathijs Sanders, Livio Trusolino, Andrea Bertotti, Isidro Cortes-Ciriano, Mathew Garnett. MSI cancer associated DNA (TA)n-dinucleotide repeat expansions and implications for Werner synthetic lethality. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4327.

Extrachromosomal Amplification of Human Papillomavirus Episomes Is a Mechanism of Cervical Carcinogenesis.

Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

Long-Read Whole-Genome Sequencing Using a Nanopore Sequencer and Detection of Structural Variants in Cancer Genomes.

Long-read sequencing technologies enable us to precisely identify structural variants (SVs), which would be occasionally associated with various types of diseases, including cancers. In this section, we introduce experimental and computational procedures for conducting long-read whole-genome sequencing (WGS) of cancer genomes from fresh frozen tissues/cells. We also demonstrate the analysis of SVs in cancer genomes using long-read WGS data from lung cancer cell lines by several representative computational tools, such as cuteSV and Sniffles2, as examples.

Validation and Application of Long-Read Whole-Genome Sequencing for Antimicrobial Resistance Gene Detection and Antimicrobial Susceptibility Testing.

Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing. We sought to identify best practices in ONT sequencing protocols. As some variability in sequencing results may be introduced by the DNA extraction methodology, we tested three DNA extraction kits across three independent laboratories using a representative set of six bacterial isolates to investigate accuracy and reproducibility of ONT technology. All DNA extraction techniques showed comparable performance; however, the DNeasy PowerSoil Pro kit had the highest sequencing yield. This kit was subsequently applied to 42 sequentially collected bacterial isolates from blood cultures to assess Ares Genetics's pipelines for predictive whole-genome sequencing antimicrobial susceptibility testing (WGS-AST) performance compared to phenotypic triplicate broth microdilution results. WGS-AST results ranged across the organisms and resulted in an overall categorical agreement of 95% for penicillins, 82.4% for cephalosporins, 76.7% for carbapenems, 86.9% for fluoroquinolones, and 96.2% for aminoglycosides. Very major errors/major errors were 0%/16.7% (penicillins), 11.7%/3.6% (cephalosporins), 0%/24.4% (carbapenems), 2.5%/7.7% (fluoroquinolones), and 0%/4.1% (aminoglycosides), respectively. This work showed that, although additional refinements are necessary, ONT sequencing demonstrates potential as a method to perform WGS-AST on cultured isolates for patient care.

125. Induction of Resistance Against Antipseudomonal Agents: Comparison of Novel b-Lactam/b-Lactamase Inhibitor (BL/BLI) Combinations and Other b-lactam Agents

Abstract Background The acquisition of mutations is the main driver of β-lactam resistance in Pseudomonas aeruginosa isolates. New BL/BLIs, such as ceftazidime-avibactam (CAZ-AVI), ceftolozane-tazobactam (C/T), and imipenem-relebactam (IMI-REL), are active against most P. aeruginosa isolates from US hospitals, but the ability of these agents to induce resistance have not been explored. We subjected 8 P. aeruginosa isolates, including ATCC 27853 and 7 clinical isolates, to a 10-day serial passage with 6 antipseudomonal agents to evaluate resistance levels and mechanisms in terminal mutant strains. Fold change in MIC results from parent isolate to terminal mutant Methods Serial passaging was performed in broth microdilution (BMD) for CAZ-AVI, IMI-REL, C/T, meropenem (MEM), cefepime (FEP), and piperacillin-tazobactam (P/T). The MIC of the terminal mutants was determined after 2X passaging on drug-free agar. Parent strains and terminal mutants were subjected to short-read whole genome sequencing (WGS) at 100X coverage. Parent isolates were sequenced using long-read WGS and the data was combined with short-reading sequencing for single nucleotide polymorphism (SNP) analysis. Results Overall, IMI-REL (1- to 4-fold) and CAZ-AVI (2- to 8-fold) displayed lower fold increases in MIC values when compared to other agents tested (Figure). Of the CAZ-AVI terminal mutants, 3 displayed a nalD regulator alteration, and 1 of these had a clpA chaperone missense substitution. FEP terminal mutants exhibited alterations in ampD, mexB, and the TetR family transcriptional regulator AmrR. C/T mutants had ampG and ftsI missense alterations. MEM mutants had nalC, ftsI, and phoP missense alterations. Mutations in merR, nalC, and ampD were observed in the P/T terminal mutants. Among 2 IMI-REL terminal mutants displaying a SNP alteration, 1 displayed a nonsense mutation in pilF, a pilus forming protein. Many terminal mutants displayed alterations in genes not commonly associated to β-lactam resistance. Conclusion MEM, FEP, and P/T terminal mutants displayed high MIC values compared to those obtained after exposure to C/T, CAZ-AVI, and IMI-REL. This data might indicate a benefit of using these newer agents to prevent the emergence of high-level resistance. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support Timothy Doyle, MS, AbbVie: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Jessica Ewald, PhD, AbbVie: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support.

Experimental Approaches to Study Somatic Transposition in Drosophila Using Whole-Genome DNA Sequencing.

The extent of transposable element (TE) mobilization in different somatic tissues and throughout diverse species is not well understood. Somatic transposition is often challenging to study as it generates de novo TE insertions that represent rare genetic variants present in heterogenous tissues. Here, we describe experimental approaches that can be applied to address TE mobility in somatic tissues with the use of short- and long-read whole-genome DNA sequencing. Focusing on the analysis of the Drosophilamelanogaster intestinal and head tissues, we provide instructions on how to design, perform, and validate experiments that aim at detecting somatic transposition. In addition to providing examples of protocols, this chapter intends to deliver general experimental guidelines that may be adapted to other flytissues or to other species.

From Samples to Germline and Somatic Sequence Variation: A Focus on Next-Generation Sequencing in Melanoma Research.

Next-generation sequencing (NGS) applications have flourished in the last decade, permitting the identification of cancer driver genes and profoundly expanding the possibilities of genomic studies of cancer, including melanoma. Here we aimed to present a technical review across many of the methodological approaches brought by the use of NGS applications with a focus on assessing germline and somatic sequence variation. We provide cautionary notes and discuss key technical details involved in library preparation, the most common problems with the samples, and guidance to circumvent them. We also provide an overview of the sequence-based methods for cancer genomics, exposing the pros and cons of targeted sequencing vs. exome or whole-genome sequencing (WGS), the fundamentals of the most common commercial platforms, and a comparison of throughputs and key applications. Details of the steps and the main software involved in the bioinformatics processing of the sequencing results, from preprocessing to variant prioritization and filtering, are also provided in the context of the full spectrum of genetic variation (SNVs, indels, CNVs, structural variation, and gene fusions). Finally, we put the emphasis on selected bioinformatic pipelines behind (a) short-read WGS identification of small germline and somatic variants, (b) detection of gene fusions from transcriptomes, and (c) de novo assembly of genomes from long-read WGS data. Overall, we provide comprehensive guidance across the main methodological procedures involved in obtaining sequencing results for the most common short- and long-read NGS platforms, highlighting key applications in melanoma research.

Genome-Wide Methylation and Haplotype-Resolved Aberrant Somatic Hypermutation Patterns in B-Cell Lymphomas

Introduction Diffuse large B-cell lymphoma (DLBCL) is a genetically, phenotypically, and clinically heterogenous disease. Multiple molecular classification systems using either genomic aberrations or gene expression profiles are used to delineate subgroups with shared biology. The cell of origin (COO) gene expression-based classification system stratifies DLBCLs into germinal center B-cell-like (GCB) and activated B-cell-like subgroups of DLBCL. More recently, the double-hit signature (DHITsig) has been used to further stratify GCB DLBCLs into DHITsig+ and DHITsig-, where the former is characterized by a gene expression profile reminiscent of the GC dark zone and inferior outcomes. Currently, patterns of somatic driver mutations can only partially explain the expression of DHITsig in some tumors. Genetic features such as DNA methylation have been shown to be associated with transcriptional repression and altered chromatin structure. We hypothesized that resolving DNA methylation patterns genome-wide using long read whole genome sequencing (LR-WGS) may reveal epigenetic differences associated with differential gene expression profiles within the DHITsig+ and DHITsig- subgroups and in the context of histologic transformation from follicular lymphoma. Methods Whole genome sequencing data of 10 patient samples and 4 cell line GCB DLBCL samples were generated using both short read (SR-WGS; Illumina) and LR-WGS (Oxford Nanopore Technologies, PromethION). The complementary approach combines somatic variant calling from highly accurate SR data with DNA methylation calling from LR data. GCB samples were selected based on the NanoString DLBCL90 assay and further subdivided into DHITsig+ (N=4) and DHITsig- (N=3). RNA sequencing data of 9 patient samples and 3 GCB DLBCL cell lines were generated and analyzed using Salmon and DESeq2. Simple somatic mutations from SR-WGS data were called using an ensemble of somatic variant callers, which confirmed all cases to belong to the EZB genetic subgroup. Methylated bases were called with Nanopolish followed by a differential methylation analysis using the DSS package in R to identify differences in methylation between both subgroups. Differentially methylated regions (DMRs) were annotated for GC B-cell specific superenhancers, CpG islands, and genes. Using WhatsHap and Nanomethphase, heterozygous single nucleotide polymorphisms (SNPs) from SR and LR data were used to phase LRs into haplotypes. Haplotype analyses were performed to determine the phase of aberrant somatic hypermutation (aSHM) and methylation patterns in the context of proximal translocation breakpoints. Results Most DMRs were found to be associated with regulatory regions such as promoters, superenhancers, and CpG islands. We found methylation differences between DHITsig+ and DHITsig- subgroups in 1430 superenhancers known to be active in GC B-cells. Within these superenhancer regions, DHITsig+ tumors were typically hypomethylated relative to DHITsig-. Several DHITsig classifier genes, including WNK2, TNFSF8, and IL21R, had methylation patterns inverted relative to the typical expression pattern (i.e. low methylation with high expression). Similarly, among 633 differentially methylated promoters, several including TCF3 and WNK2 demonstrated the same inverted methylation pattern. CpG islands showed distinct patterns of methylation in both subgroups that were inversely associated with expression, this also included genes differentially expressed between DHITsig+ and DHITsig- tumors. The DHITsig- group had increased levels of methylation of DHITsig genes in both promoters and gene bodies suggesting a functional role for gene body methylation. Finally, we observed that a high density of mutations near the BCL2 transcription start site consistently phased into the same haplotype as the BCL2-IGH translocation in five tumours, demonstrating the allele specificity of aSHM associated with these events (Panel B). Conclusions These results demonstrate differences in methylation between the subgroups. The association of hypomethylated superenhancers, promoters, and CpG islands with genes over-expressed in DHITsig+ suggests these methylation patterns may contribute to the DHITsig phenotype. These results may aid in improving GCB DLBCL patient classification and reveal factors contributing to its heterogeneity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-read sequence analysis for clustered genomic copy number aberrations revealed architectures of intricately intertwined rearrangements.

Most chromosomal aberrations revealed by chromosomal microarray testing (CMA) are simple; however, very complex chromosomal structural rearrangements can also be found. Although the mechanism of structural rearrangements has been gradually revealed, not all mechanisms have been elucidated. We analyzed the breakpoint-junctions (BJs) of two or more clustered copy number variations (CNVs) in the same chromosome arms to understand their conformation and the mechanism of complex structural rearrangements. Combining CMA with long-read whole-genome sequencing (WGS) analysis, we successfully determined all BJs for the clustered CNVs identified in four patients. Multiple CNVs were intricately intertwined with each other, and clustered CNVs in four patients were involved in global complex chromosomal rearrangements. The BJs of two clustered deletions identified in two patients showed microhomologies, and their characteristics were explained by chromothripsis. In contrast, the BJs in the other two patients, who showed clustered deletions and duplications, consisted of blunt-end and nontemplated insertions. These findings could be explained only by alternative nonhomologous end-joining, a mechanism related to polymerase theta. All the patients had at least one inverted segment. Three patients showed cryptic aberrations involving a disruption and a deletion/duplication, which were not detected by CMA but were first identified by WGS. This result suggested that complex rearrangements should be considered if clustered CNVs are observed in the same chromosome arms. Because CMA has potential limitations in genotype-phenotype correlation analysis, a more detailed analysis by whole genome examination is recommended in cases of suspected complex structural aberrations.

Robust Genetic Analysis of the X-Linked Anophthalmic (Ie) Mouse.

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000–58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism.

Large-Scale Sequencing of Borreliaceae for the Construction of Pan-Genomic-Based Diagnostics.

The acceleration of climate change has been associated with an alarming increase in the prevalence and geographic range of tick-borne diseases (TBD), many of which have severe and long-lasting effects—particularly when treatment is delayed principally due to inadequate diagnostics and lack of physician suspicion. Moreover, there is a paucity of treatment options for many TBDs that are complicated by diagnostic limitations for correctly identifying the offending pathogens. This review will focus on the biology, disease pathology, and detection methodologies used for the Borreliaceae family which includes the Lyme disease agent Borreliella burgdorferi. Previous work revealed that Borreliaceae genomes differ from most bacteria in that they are composed of large numbers of replicons, both linear and circular, with the main chromosome being the linear with telomeric-like termini. While these findings are novel, additional gene-specific analyses of each class of these multiple replicons are needed to better understand their respective roles in metabolism and pathogenesis of these enigmatic spirochetes. Historically, such studies were challenging due to a dearth of both analytic tools and a sufficient number of high-fidelity genomes among the various taxa within this family as a whole to provide for discriminative and functional genomic studies. Recent advances in long-read whole-genome sequencing, comparative genomics, and machine-learning have provided the tools to better understand the fundamental biology and phylogeny of these genomically-complex pathogens while also providing the data for the development of improved diagnostics and therapeutics.

First detection and origin of multi-drug resistant Klebsiella pneumoniae ST15 harboring OXA-48 in South America

The emergence and spread of carbapenem resistant clones is of major concern for global health. This study aimed to characterize the first detected Klebsiella pneumoniae ST15 harboring the epidemic carbapenemase OXA-48 in South America. During a routine colonization screening with carbapenem-resistant bacteria, one K. pneumoniae strain (CGHM01) was isolated from the urine of a hospitalized patient suffering from a neurodegenerative disease in Uruguay. We used long-read whole-genome sequencing and a phylogenomic approach to characterize the emergence of K. pneumoniae CGHM01. K. pneumoniae CGHM01 is a multi-drug resistant strain carrying an IncL/M plasmid that encodes the carbapenemase gene blaOXA-48 within the Tn1999.2 transposon. Also, it carries an IncR plasmid harboring a class I integron with an array of antibiotic resistance genes including the extended-spectrum beta-lactamase blaCTX-M-15. Two copies of blaCTX-M-15 were also inserted in different positions of the chromosome. CGHM01 belongs to a ST15 sublineage that likely originated in continental Spain around 2012. The asymptomatic carriage of this strain in the urinary tract warns of difficulties for detection and reporting of emerging carbapenem-resistant clones in new geographic areas where these are not endemic.