Combination Of Whole-genome Sequencing Research Articles

Genomic Characterization of Carbapenemase-Producing Enterobacter hormaechei, Serratia marcescens, Citrobacter freundii, Providencia stuartii, and Morganella morganii Clinical Isolates from Bulgaria.

Carbapenemase-producing Enterobacter spp. Serratia marcescens, Citrobacter freundii, Providencia spp., and Morganella morganii (CP-ESCPM) are increasingly identified as causative agents of nosocomial infections but are still not under systematic genomic surveillance. In this study, using a combination of whole-genome sequencing and conjugation experiments, we sought to elucidate the genomic characteristics and transferability of resistance genes in clinical CP-ESCPM isolates from Bulgaria. Among the 36 sequenced isolates, NDM-1 (12/36), VIM-4 (11/36), VIM-86 (8/36), and OXA-48 (7/36) carbapenemases were identified; two isolates carried both NDM-1 and VIM-86. The majority of carbapenemase genes were found on self-conjugative plasmids. IncL plasmids were responsible for the spread of OXA-48 among E. hormaechei, C. freundii, and S. marcescens. IncM2 plasmids were generally associated with the spread of NDM-1 in C. freundii and S. marcescens, and also of VIM-4 in C. freundii. IncC plasmids were involved in the spread of the recently described VIM-86 in P. stuartii isolates. IncC plasmids carrying blaNDM-1 and blaVIM-86 were observed too. blaNDM-1 was also detected on IncX3 in S. marcescens and on IncT plasmid in M. morganii. The significant resistance transfer rates we observed highlight the role of the ESCPM group as a reservoir of resistance determinants and stress the need for strengthening infection control measures.

Anaerolentibacter hominis gen. nov. sp. nov., Diplocloster hominis sp. nov. and Pilosibacter fragilis gen. nov. sp. nov., isolated from human faeces.

Three Gram-positive, obligately anaerobic bacterial strains, namely CSJ-1T, CSJ-3T, and CSJ-4T, were isolated from faeces of healthy persons. They were characterized through a combination of whole-genome sequencing, phenotypic traits, and metabolomic analysis. The genome sizes of CSJ-1T, CSJ-4T, and CSJ-3T were 3.3, 3.8, and 6.1 Mbp, with DNA G+C contents of 47.2, 48.3, and 48.8 mol%, respectively. Strain CSJ-3T was identified as representing a novel species, Diplocloster hominis (type strain CSJ-3T=CGMCC 1.18033T=JCM 36512T) of the genus Diplocloster. The 16S rRNA gene sequence similarity and whole genome average nucleotide identity (gANI) of CSJ-4T to its closest related species, Diplocloster modestus ASD 4241T, were 98.3 and 91.4 %, respectively. Comparative analysis of 16S rRNA gene sequences showed 91.6 % similarity between CSJ-1T and its closest phylogenetic neighbour, Catenibacillus scindens DSM 106146T, and 93.3 % similarity between CSJ-4T and its closest relative strain, Clostridium fessum SNUG30386T. Based on the polyphasic taxonomic results, we proposed two novel genera and three novel species. Strain CSJ-1T was identified as representing a novel species of novel genus, Anaerolentibacter hominis gen. nov. sp. nov. (type strain CSJ-1T=CGMCC 1.18046T=JCM 36511T) of the family Lachnospiraceae, and strain CSJ-4T was identified as representing a novel species of novel genus Pilosibacter fragilis gen. nov. sp. nov. (type strain CSJ-4T=CGMCC 1.18026T= JCM 36513T) of the family Clostridiaceae.

Comparison of contemporary invasive and non-invasive Streptococcus pneumoniae isolates reveals new insights into circulating anti-microbial resistance determinants.

Streptococcus pneumoniae is a major human pathogen with a high burden of disease. Non-invasive isolates (those found in non-sterile sites) are thought to be a key source of invasive isolates (those found in sterile sites) and a reservoir of anti-microbial resistance (AMR) determinants. Despite this, pneumococcal surveillance has almost exclusively focused on invasive isolates. We aimed to compare contemporaneous invasive and non-invasive isolate populations to understand how they interact and identify differences in AMR gene distribution. We used a combination of whole-genome sequencing and phenotypic anti-microbial susceptibility testing and a data set of invasive (n = 1,288) and non-invasive (n = 186) pneumococcal isolates, collected in Victoria, Australia, between 2018 and 2022. The non-invasive population had increased levels of antibiotic resistance to multiple classes of antibiotics including beta-lactam antibiotics penicillin and ceftriaxone. We identified genomic intersections between the invasive and non-invasive populations and no distinct phylogenetic clustering of the two populations. However, this analysis revealed sub-populations overrepresented in each population. The sub-populations that had high levels of AMR were overrepresented in the non-invasive population. We determined that WamR-Pneumo was the most accurate in silico tool for predicting resistance to the antibiotics tested. This tool was then used to assess the allelic diversity of the penicillin-binding protein genes, which acquire mutations leading to beta-lactam antibiotic resistance, and found that they were highly conserved (≥80% shared) between the two populations. These findings show the potential of non-invasive isolates to serve as reservoirs of AMR determinants.

BNT162b2 vaccine protection against omicron and effect of previous infection variant and vaccination sequence among children and adolescents in Singapore: a population-based cohort study.

Information on variant-specific vaccine protection and the effect of previous infection variant is scarce in children. We aimed to ascertain the level of protection conferred by BNT162b2 COVID-19 vaccination against omicron variant infection (BA.4 or BA.5, and XBB) in a previously infected national paediatric cohort. We also explored the association between sequence of previous infection (variant) and vaccination on protection. We did a retrospective, population-based cohort study using the national databases of all confirmed SARS-CoV-2 infections, vaccines administered, and demographic records maintained by the Ministry of Health, Singapore. The study cohort consisted of children aged 5-11 years and adolescents aged 12-17 years who had a previous SARS-CoV-2 infection from Jan 1, 2020, to Dec 15, 2022. People who were infected during the pre-delta period or were immunocompromised (received three vaccination doses [children 5-11 years old] and four vaccinations doses [adolescents 12-17 years old]) were excluded. Those who had multiple episodes of infection before the study start date, were not vaccinated before infection but completed three doses, received bivalent mRNA vaccine, or received non-mRNA vaccine doses were also excluded. All SARS-CoV-2 infections confirmed by reverse transcriptase polymerase chain reaction or rapid antigen testing were grouped into delta, BA.1, BA.2, BA.4 or BA.5, or XBB variants using a combination of whole-genome sequencing, S-gene target failure results, and imputation. For BA.4 or BA.5, the study outcome period was June 1-Sept 30, 2022, and for XBB variants the outcome period was Oct 18-Dec 15, 2022. Incidence rate ratios between vaccinated and unvaccinated were derived using adjusted Poisson regressions and vaccine effectiveness was estimated as (1-risk ratio) × 100%. 135 197 people aged 5-17 years (79 332 children and 55 865 adolescents) were included in the cohort for the vaccine effectiveness analysis against omicron BA.4 or BA.5, and 164 704 people aged 5-17 years (97 235 children and 67 469 adolescents) were included for the analysis against omicron XBB. Approximately 47% of participants were female and 53% were male. Among those previously infected, vaccine effectiveness against BA.4 or BA.5 infection in fully vaccinated children (two doses) was 74·0% (95% CI 67·7-79·1) and in adolescents (three doses) was 85·7% (80·2-89·6). Against XBB, protection conferred with full vaccination was lower at 62·8% (95% CI 42·3-76·0) in children and 47·9% (20·2-66·1) in adolescents. In children, receipt of two-dose vaccination before first SARS-CoV-2 infection provided them with the highest protection against subsequent BA.4 or BA.5 infection at 85·3% (95% CI 80·2-89·1); however, this was not shown to be the case for adolescents. First infection variant had an effect on vaccine effectiveness against omicron BA.4 or BA.5 reinfection in the following descending order: BA.2 conferred the highest protection (92·3% [95% CI 88·9-94·7] in children and 96·4% [93·5-98·0] in adolescents) followed by BA.1 (81·9% [75·9-86·4] in children and 95·0% [91·6-97·0] in adolescents), and delta which conferred the lowest protection (51·9% [5·3-75·6] in children and 77·5% [63·9-86·0] in adolescents). In previously infected children and adolescents, BNT162b2 vaccination provided additional protection against omicron BA.4 or BA.5 and XBB variants compared with those who remained unvaccinated. Hybrid immunity against XBB was lower than against BA.4 or BA.5, especially in adolescents. Early vaccination of previously uninfected children before their first SARS-CoV-2 exposure could potentially strengthen population immunity resilience against future variants. None.

Glyoxylate Shunt and Pyruvate-to-Acetoin Shift Are Specific Stress Responses Induced by Colistin and Ceragenin CSA-13 in Enterobacter hormaechei ST89.

Ceragenins, including CSA-13, are cationic antimicrobials that target the bacterial cell envelope differently than colistin. However, the molecular basis of their action is not fully understood. Here, we examined the genomic and transcriptome responses by Enterobacter hormaechei after prolonged exposure to either CSA-13 or colistin. Resistance of the E. hormaechei 4236 strain (sequence type 89 [ST89]) to colistin and CSA-13 was induced in vitro during serial passages with sublethal doses of tested agents. The genomic and metabolic profiles of the tested isolates were characterized using a combination of whole-genome sequencing (WGS) and transcriptome sequencing (RNA-seq), followed by metabolic mapping of differentially expressed genes using Pathway Tools software. The exposure of E. hormaechei to colistin resulted in the deletion of the mgrB gene, whereas CSA-13 disrupted the genes encoding an outer membrane protein C and transcriptional regulator SmvR. Both compounds upregulated several colistin-resistant genes, such as the arnABCDEF operon and pagE, including genes coding for DedA proteins. The latter proteins, along with beta-barrel protein YfaZ and VirK/YbjX family proteins, were the top overexpressed cell envelope proteins. Furthermore, the l-arginine biosynthesis pathway and putrescine-ornithine antiporter PotE were downregulated in both transcriptomes. In contrast, the expression of two pyruvate transporters (YhjX and YjiY) and genes involved in pyruvate metabolism, as well as genes involved in generating proton motive force (PMF), was antimicrobial specific. Despite the similarity of the cell envelope transcriptomes, distinctly remodeled carbon metabolism (i.e., toward fermentation of pyruvate to acetoin [colistin] and to the glyoxylate pathway [CSA-13]) distinguished both antimicrobials, which possibly reflects the intensity of the stress exerted by both agents. IMPORTANCE Colistin and ceragenins, like CSA-13, are cationic antimicrobials that disrupt the bacterial cell envelope through different mechanisms. Here, we examined the genomic and transcriptome changes in Enterobacter hormaechei ST89, an emerging hospital pathogen, after prolonged exposure to these agents to identify potential resistance mechanisms. Interestingly, we observed downregulation of genes associated with acid stress response as well as distinct dysregulation of genes involved in carbon metabolism, resulting in a switch from pyruvate fermentation to acetoin (colistin) and the glyoxylate pathway (CSA-13). Therefore, we hypothesize that repression of the acid stress response, which alkalinizes cytoplasmic pH and, in turn, suppresses resistance to cationic antimicrobials, could be interpreted as an adaptation that prevents alkalinization of cytoplasmic pH in emergencies induced by colistin and CSA-13. Consequently, this alteration critical for cell physiology must be compensated via remodeling carbon and/or amino acid metabolism to limit acidic by-product production.

Novel ITPA variants identified by whole genome sequencing and RNA sequencing.

Approximately 80% of rare diseases have a genetic cause, and an accurate genetic diagnosis is necessary for disease management, prognosis prediction, and genetic counseling. Whole-exome sequencing (WES) is a cost-effective approach for exploring the genetic cause, but several cases often remain undiagnosed. We combined whole genome sequencing (WGS) and RNA sequencing (RNA-seq) to identify the pathogenic variants in an unsolved case using WES. RNA-seq revealed aberrant exon 4 and exon 6 splicing of ITPA. WGS showed a previously unreported splicing donor variant, c.263+1G>A, and a novel heterozygous deletion, including exon 6. Detailed examination of the breakpoint indicated the deletion caused by recombination between Alu elements in different introns. The proband was found to have developmental and epileptic encephalopathies caused by variants in the ITPA gene. The combination of WGS and RNA-seq may be effective in diagnosing conditions in proband who could not be diagnosed using WES.

Characterization of a novel polyextremotolerant fungus, Exophiala viscosa, with insights into its melanin regulation and ecological niche.

Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primar yeast form. These fungi grow in xeric, nutrient depletes environments which implies that they require highly flexible metabolisms and have been suggested to contain the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community are not well understood. We have isolated 2 novel black yeasts from the genus Exophiala that were recovered from dryland biological soil crusts. Despite notable differences in colony and cellular morphology, both fungi appear to be members of the same species, which has been named Exophiala viscosa (i.e. E. viscosa JF 03-3 Goopy and E. viscosa JF 03-4F Slimy). A combination of whole genome sequencing, phenotypic experiments, and melanin regulation experiments have been performed on these isolates to fully characterize these fungi and help decipher their fundamental niche within the biological soil crust consortium. Our results reveal that E. viscosa is capable of utilizing a wide variety of carbon and nitrogen sources potentially derived from symbiotic microbes, can withstand many forms of abiotic stresses, and excretes melanin which can potentially provide ultraviolet resistance to the biological soil crust community. Besides the identification of a novel species within the genus Exophiala, our study also provides new insight into the regulation of melanin production in polyextremotolerant fungi.

Combination of whole genome sequencing and supervised machine learning provides unambiguous identification of eae-positive Shiga toxin-producing Escherichia coli.

The objective of this study was to develop, using a genome wide machine learning approach, an unambiguous model to predict the presence of highly pathogenic STEC in E. coli reads assemblies derived from complex samples containing potentially multiple E. coli strains. Our approach has taken into account the high genomic plasticity of E. coli and utilized the stratification of STEC and E. coli pathogroups classification based on the serotype and virulence factors to identify specific combinations of biomarkers for improved characterization of eae-positive STEC (also named EHEC for enterohemorrhagic E.coli) which are associated with bloody diarrhea and hemolytic uremic syndrome (HUS) in human. The Machine Learning (ML) approach was used in this study on a large curated dataset composed of 1,493 E. coli genome sequences and 1,178 Coding Sequences (CDS). Feature selection has been performed using eight classification algorithms, resulting in a reduction of the number of CDS to six. From this reduced dataset, the eight ML models were trained with hyper-parameter tuning and cross-validation steps. It is remarkable that only using these six genes, EHEC can be clearly identified from E. coli read assemblies obtained from in silico mixtures and complex samples such as milk metagenomes. These various combinations of discriminative biomarkers can be implemented as novel marker genes for the unambiguous EHEC characterization from different E. coli strains mixtures as well as from raw milk metagenomes.

Abstract 3517: Multi-modal single-cell and spatial genomics reveals genomic, adaptive and microenvironmental features of human non-small cell lung cancer brain metastasis

Abstract Non-small cell lung cancer (NSCLC) accounts for nearly half of all newly diagnosed patients with brain metastasis (BM), followed by melanoma and breast carcinomas. The presence of BM is associated with reduced response to several modern cancer therapies and a poor prognosis, but the underlying molecular underpinnings remain poorly understood. Here, we performed multi-modal single-nucleus RNA, T cell receptor, single-cell spatial, and whole-genome sequencing (WGS) of 44 primary NSCLC tumors (PTs) and BMs. Through combination of WGS with inferred copy-number alterations (CNAs) and gene expression snRNA-seq, we robustly identify malignant cells despite the presence of healthy cell mosaicism. We find a strong association of chromosomal instability (CIN) and brain-metastatic organotropism. Through integration with clinical information and thousands of publicly available whole-exome sequencing (WES) profiles obtained from patients with NSCLC, we validate this observation and show that CIN progressively increases from PTs to extracranial metastases (ECMs) and is the highest in BMs. Using non-negative matrix factorization, we identify recurrent transcriptional hallmarks cancer metastasis, and additionally find that cancer cells from BMs strongly enrich for a neuronal-like cell state. At single-cell resolution, we indeed identify a rare cancer cell population genomically define by very high CIN, and transcriptionally characterized by a program of epithelial-to-mesenchymal transition (EMT), neuronal-like differentiation, and loss of lineage attribution. We show in our data and external scRNA-seq data that this cell state does not ecist in healthy lungs, progressively enriches from PTs to ECMs, and is most abundant in BMs, suggesting that these cells may indeed give rise to BMs. Furthermore, through integration of snRNA/TCR-seq and spatial transcriptomics, we find distinct tumor-microenvironments across disease sites, including, nearly exclusive expansion of tissue-resident myeloid cells in PTs, while BMs are largely dominated by dense infiltration with monocyte-derived macrophages and granulocytes, impaired T cell infiltration, activation and clonal expansion. Lastly, spatial transcriptomics also recurrent, cell-type specific patterns of geographic variability in key pathways, including antigen presentation, EMT, oxidative phosphorylation, and inflammatory response and associated cellular micro-niches. Together, this work identifies cellular, genomic, and transcriptional features of NSCLC BMs and has important therapeutic implications for novel therapies, in particular immunomodulatory approaches targeting cell types/states unique to disease sites. Citation Format: Somnath Tagore, Lindsay Caprio, Amit Dipak Amin, Irving Barrera, Johannes Melms, Karan Luthria, Yiping Wang, Yohanna Georgis, Abhi Jaiswal, Galina G. Lagos, Zachary Walsh, Parin Shah, Jana Biermann, Neha Sheikh, Priyanka Ramaradj, Niroshana Anandasabapathy, Hanina Hibshoosh, Gary Schwartz, Brian Henick, Alison Taylor, Fei Chen, Benjamin Izar. Multi-modal single-cell and spatial genomics reveals genomic, adaptive and microenvironmental features of human non-small cell lung cancer brain metastasis [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 3517.

Genomic Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Among University Students in Western Pennsylvania.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control on college campuses is challenging given communal living and student social dynamics. Understanding SARS-CoV-2 transmission among college students is important for the development of optimal control strategies. SARS-CoV-2 nasal swab samples were collected from University of Pittsburgh students for symptomatic testing and asymptomatic surveillance from August 2020 through April 2021 from 3 campuses. Whole-genome sequencing (WGS) was performed on 308 samples, and contact tracing information collected from students was used to identify transmission clusters. We identified 31 Pangolin lineages of SARS-CoV-2, the majority belonging to B.1.1.7 (Alpha) and B.1.2 lineages. Contact tracing identified 142 students (46%) clustering with each other; WGS identified 53 putative transmission clusters involving 216 students (70%). WGS identified transmissions that were missed by contact tracing. However, 84 cases (27%) could not be linked by either WGS or contact tracing. Clusters were most frequently linked to students residing in the same dormitory, off-campus roommates, friends, or athletic activities. The majority of SARS-CoV-2-positive samples clustered by WGS, indicating significant transmission across campuses. The combination of WGS and contact tracing maximized the identification of SARS-CoV-2 transmission on campus. WGS can be used as a strategy to mitigate, and further prevent transmission among students.

Long-Term Clonal Inversion in an MDS-RS Case with Dual SF3B1 Mutations

Recent large-scale studies have traced SF3B1 mutations as a driving event in clonal establishment and expansion, and a hallmark of myelodysplastic syndrome with ring sideroblasts (MDS-RS). However, the mechanisms underlying the fitness advantage allowing SF3B1-mutant (SF3B1mt) clones to thrive remain poorly understood. Thus, tracking these events in conditions of low noise is crucial to our comprehension of clonal hematopoiesis. Here we report a 12-year clinical and molecular follow-up of a unique dual SF3B1mt MDS-RS case displaying a dynamic state of clonal competition between two clones propagated by a N626D and K666N mutation, respectively (Panel A). After an initial rapid expansion of the N626D clone compared to the relatively stable K666N clone during the initial follow-up period of ~2 years, the K666N clone spontaneously expanded at expense of the N626D clone to eventually dominate the bone marrow, driving a complete inversion of clonal prevalence. Clinically, these active shifts of high clonal involvement in the bone marrow were accompanied by stable subclinical anemia (median 12.5 g/L, range 12.0 - 13.2 g/L) and a continued decrease in the ring sideroblast phenotype over time (from 42% to 8% RS) in the absence of any treatment. The stability of this patient's clinical profile was therefore considered as unlikely to confound further characterization. Through a combination of whole-genome sequencing (WGS) of hematopoietic stem and progenitor cell (HSPC)-generated colony-forming units (CFU), combined DNA + RNAseq (TargetSeq) of FACS-purified hematopoietic stem cells (HSCs) and megakaryocyte-erythroid progenitor cells (MEP), and high-throughput 3'-based 10X Genomics single-cell RNAseq of CD34+ HSPCs, we molecularly characterized the two distinct SF3B1mt clones in bone marrow samples acquired before, during and after the points of clonal inversion (Panel B). Using WGS of single cell-derived colonies, we established that the acquisition of either the N626D or K666N mutation had preceded the diagnosis of MDS-RS by at least 20 years. Moreover, both mutations were associated with an increased mutational rate as compared to wildtype colonies, and the two clones presented distinct patterns of additional coding mutations as potential co-drivers of expansion. Mirroring previous characterizations of SF3B1mt HSPCs, we identified a common and deleterious alternative splicing profile to both genotypes via TargetSeq which included mis-splicing events in the known SF3B1mt targets MAP3K7 and SEPTIN6. In addition, we identified mutation-specific splicing profiles as potentially responsible for distinct downstream distinctions between both clones. Through this analysis, we identified the K666N-specific loss of expression of the tumor suppressors HOXA5, RGCC and NEURL1 as potential compound factors to the known higher risk underlying SF3B1K666N mutations. Finally, we disentangled clone-driven and aging-driven transcriptomic changes to identify a significant decrease of the early hematopoietic controllers ID1, ID2 and ID3 as a potential mechanism of aging-related cellular senescence. In conclusion, this case study comprises a unique long-term follow-up of the clonal dynamics underlying two co-existing, distinct and competing SF3B1mt clones which identifies subtle molecular changes and differences underlying the expansion and progression of SF3B1mt clones. *Moura PL, Hofman IJF, Nannya Y and Aliouat A contributed equally to this work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combination of Whole Genome Sequencing and Metagenomics for Microbiological Diagnostics.

Whole genome sequencing (WGS) provides the highest resolution for genome-based species identification and can provide insight into the antimicrobial resistance and virulence potential of a single microbiological isolate during the diagnostic process. In contrast, metagenomic sequencing allows the analysis of DNA segments from multiple microorganisms within a community, either using an amplicon- or shotgun-based approach. However, WGS and shotgun metagenomic data are rarely combined, although such an approach may generate additive or synergistic information, critical for, e.g., patient management, infection control, and pathogen surveillance. To produce a combined workflow with actionable outputs, we need to understand the pre-to-post analytical process of both technologies. This will require specific databases storing interlinked sequencing and metadata, and also involves customized bioinformatic analytical pipelines. This review article will provide an overview of the critical steps and potential clinical application of combining WGS and metagenomics together for microbiological diagnosis.

Abstract 54: Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies

Abstract The use of sequencing-based assays for clinical management of pediatric cancer patients has become increasingly common. However, for many pediatric patients, gene panel based sequencing tests yield few actionable results. Given the complex genomic alterations present in many pediatric cancers, especially high-risk solid tumors, we hypothesized that an unbiased approach might reveal more actionable findings and lead to a more comprehensive understanding of these diseases. To accomplish this, we integrated whole-genome sequencing (WGS) with RNAseq in the analysis of a pediatric oncology cohort, with a focus on longitudinal cases to capture potential tumor evolution in metastatic or treated cases. Our cohort consists of 269 high-risk pediatric oncology patients, including patients with relapsed/refractory disease, metastatic disease at diagnosis, prior cancer history, a rare diagnosis, or an estimated overall survival <50%. Solid tumors, CNS tumors, and leukemia/lymphomas are all represented. In total, 391 samples were characterized using WGS (tumor ~60X; germline ~30X) and/or RNAseq (tumor, polyA selected, ≥20 million reads). For 85 of these patients, multiple samples were collected at different time points (diagnosis, resection, relapse, etc.) to identify changes in the cancer over time. If panel testing was performed as part of their clinical care, a comparison to the integrated WGS/RNA analysis was made. WGS was used to identify variants (SNVs), structural rearrangements (SVs), mutational signatures, and copy-number alterations (CNAs). RNAseq was used to identify gene expression outliers, gene fusions, and confirm the expression of variants identified using WGS. The combination of WGS and RNAseq was then used to identify and prioritize potentially actionable variants for each patient. Our results show that the integration of WGS and RNAseq can provide more and higher-quality actionable information than either modality alone, whilst also capturing the majority of actionable variants detected by panel sequencing. RNAseq identified not only druggable fusions and expression outliers, but also many rare and novel fusions. WGS provided fusion validation but highlighted the limitations of WGS alone in identifying fusions resulting from complex SVs. Conversely, WGS was adept at capturing genome-wide patterns of CNAs and loss of heterozygosity that are missed by gene-centric panels. Further RNAseq integration enabled prioritization of expressed SNVs as well as CNAs and SVs that significantly alter gene expression. We also used WGS to extract mutational signatures and tracked their evolution across longitudinal samples. We found potentially biologically significant differences in therapy-induced mutations caused by platinum and alkylating agents. Our unbiased approach has enabled further discovery that advances our understanding of these rare and highly aggressive malignancies. Citation Format: Henry J. Martell, Avanthi Tayi Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Maria V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, E. Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 54.

How adaptive laboratory evolution can boost yeast tolerance to lignocellulosic hydrolyses.

The yeast Saccharomyces cerevisiae is an excellent candidate for establishing cell factories to convert lignocellulosic biomass into chemicals and fuels. To enable this technology, yeast robustness must be improved to withstand the fermentation inhibitors (e.g., weak organic acids, phenols, and furan aldehydes) resulting from biomass pretreatment and hydrolysis. Here, we discuss how evolution experiments performed in the lab, a method commonly known as adaptive laboratory evolution (ALE), may contribute to lifting yeast tolerance against the inhibitors of lignocellulosic hydrolysates (LCHs). The key is that, through the combination of whole-genome sequencing and reverse engineering, ALE provides a robust platform for discovering and testing adaptive alleles, allowing to explore the genetic underpinnings of yeast responses to LCHs. We review the insights gained from past evolution experiments with S. cerevisiae in LCH inhibitors and propose experimental designs to optimise the discovery of genetic variants adaptive to biomass toxicity. The knowledge gathered through ALE projects is envisaged as a roadmap to engineer superior yeast strains for biomass-based bioprocesses.

Insights into the Complexity of a Dormant Mycobacterium tuberculosis Cluster Once Transmission Is Resumed.

ABSTRACTGenotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spanish-born individuals. In one case, two Mycobacterium tuberculosis clonal variants were identified. We reanalyzed the cluster, supported by the characterization of multiple cultured isolates and respiratory specimens, whole-genome sequencing, and epidemiological case interviews. Our findings showed that the cluster, which was initially thought to have restarted activity with just a single case harboring a small degree of within-host diversity, was in fact currently growing due to coincidental reactivation of past exposures, with clonal diversity transmitted throughout the cluster. In one case, within-host diversity was amplified, probably due to prolonged diagnostic delay.IMPORTANCE The precise study of the dynamics of tuberculosis transmission in socio-epidemiologically complex scenarios may require more thorough analysis than the standard molecular epidemiology strategies. Our study illustrates the epidemiological and bacteriological complexity present in a transmission cluster in a challenging epidemiological setting with a high proportion of migrant cases. The combination of whole-genome sequencing, refined and refocused epidemiological interviews, and in-depth analysis of the bacterial composition of sputa and cultured isolates was crucial in order to correctly reinterpret the true nature of this cluster. Our global approach allowed us to reinterpret correctly the unnoticed epidemiological and bacteriological complexity involved in the Mycobacterium tuberculosis transmission event under study, which had been overlooked by the usual molecular epidemiology approaches.

Regional dissemination of carbapenem-resistant Enterobacteriaceae accompanying with enhanced resistance in Northern Osaka, Japan

With the rapid spread of multidrug-resistant bacteria, carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide as a major concern because of limited treatment options. Carbapenem resistance is mainly due to carbapenem-ase, a carbapenem-degrading enzyme, which is mainly encoded on a plasmid to spread across bacterial species. However, there have been only small-scale attempts to determine the similarities or accommodations of the plasmids disseminating regionwide. We analysed the 230 CRE isolates carrying blaIMP from 43 medical facilities in the northern Osaka area focusing on the plasmids, the main carriers of the drug resistance genes. Combination of whole genome sequencing and Southern blotting revealed the predominant dissemination of blaIMP-6 by the pKPI-6 plasmid among genetically distinct isolates, as well as the emergences of derivatives that acquired various advantages. We iden-tified heteroresistance likely causing stealth transmissions, which was generated by the transcriptional regu-lation of blaIMP-6, stabilization of blaIMP-6 through chromosomal integration, enhanced carbapenem resistance through plasmid multimerization, or broadened antimicrobial resistance due to a single point mutation in blaIMP-6. In this article, I dis-cussed the mechanisms of regional spread of CRE and enhancement of carbapenem resistance providing the insights to prevent their disseminations.

Benchmarking of Whole Genome Sequencing (WGS) and Whole Transcriptome Sequencing (WTS) As Diagnostic Tools for the Genetic Characterization of Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) in Adults

Background: In AML and ALL the application of WHO classification and ELN guidelines requires a combination of cytogenetics and targeted sequencing for specific mutations to determine the diagnostic and prognostic subgroup. WGS and WTS have emerged as comprehensive techniques that allow the simultaneous analysis and identification of all genetic alterations in a single approach with possible turnaround times of 1 week.Aim: Evaluate the accuracy of WGS and WTS in providing all relevant genetic information in a clinical setting.Patients and Methods: The cohort comprised 738 AML, 293 BCP-ALL and 124 T-ALL. The diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp) were performed on a NovaSeq instrument. Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta.Results: The combination of WGS and WTS detected all chromosomal and molecular abnormalities in the AML and ALL cohorts relevant for disease stratification and prognostication as identified by chromosome banding analysis (CBA) and targeted panel sequencing (TPS). A very high concordance between CBA and WGS was revealed for the detection of balanced structural variants (SV) with the added benefit of WGS to also detect cytogenetically cryptic rearrangements (i.e.: ETV6-MN1, NUP98-KDM5A), which all were confirmed either by FISH or RT-PCR. Fusion calling by WTS identified 96% of the WHO subtype defining rearrangements and detected 20 additional fusion transcripts relevant for disease stratification (e.g. EP300-ZNF384, TCF3-HLF) including 9 fusion transcripts that led to prognostic reassignment or could serve as a potential treatment target. Breakpoints of unbalanced SV can occur in repetitive sequences of the genome, hampering the detection by WGS. However, adding copy number alteration (CNA) calls to the analyses allows also reliable identification of unbalanced SV.WGS outperformed CBA in cases with insufficient in vitro proliferation due to suboptimal pre-analytics (i.e. longer transport time) and identified 36 chromosomal aberrations in 12 cases with CBA not evaluable. WGS's independence of in vitro cell proliferation was most impactful in ALL: 40 T-ALL cases showed a normal karyotype according to CBA. WGS detected SVs in 16 (40%) and CNAs in 20 (50%) of these cases, confirming the normal karyotype for only 9 samples. In the BCP-ALL cohort, CNV analysis identified 29 low hypodiploid and 16 high hyperdiploid karyotypes, 6 of which were missed by CBA. Due to the higher resolution and unrestricted, genome-wide assessment, WGS detected relevant gene deletions (RB1, ERG, PAX5, CDKN2A, IKZF1, ETV6, BTG1) in 59% of ALL cases, providing additional diagnostic and prognostic information. In the AML cohort CBA and WGS detected 795 CNA concordantly. In addition WGS called 54 CNA with size 1-5 MB (below the detection limit of CBA), i.e. 3 BCOR deletions in inv(3)(q21q26) cases and 67 CNA with size > 5 MB, which were missed by CBA. 35 CNA were missed by WGS due to small clone sizes (median 6% as determined by FISH). WGS detected copy neutral loss of heterozygosity (CN-LOH) in AML most frequently on 21q (n=17), 4q (n=15), 13q (n=15), 11q (n=13) and in T-ALL on 9p (n=19), mostly encompassing CDKN2A/B deletions. Expression profiling provided additional diagnostic information for 57 ALL cases (41 BCR-ABL1-like, 16 DUX4 rearranged) that can only insufficiently be obtained by WGS or CBA. WGS reliably detected all gene mutations with a VAF > 15% (n = 647) identified by TPS encompassing especially all mutations in genes relevant for WHO diagnosis and prognostication. 26/171 mutations with a VAF < 15% were missed by WGS. Evaluation of WGS data for 121 genes recurrently mutated in hematologic neoplasms revealed an additional 2 mutations per sample on average (range: 0-9) which might qualify as targets for therapy.Conclusions: WGS and WTS provide all necessary genetic information to accurately determine the diagnostic and prognostic subgroup according to WHO and ELN guidelines in AML and ALL. Compared to today's gold standards, these novel methods provide a comprehensive genome wide characterization with higher resolution that directly identifies genes of impact, offering the basis for targeted treatment selection and monitoring of residual disease. Both can be implemented with automated analysis pipelines, consequently reducing time and error rates. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Harnessing Whole Genome Sequence Data for Facility-Specific Signatures for Listeria monocytogenes: A Case Study With Turkey Processing Plants in the United States

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for the severe disease listeriosis and notorious for its ability to persist in food processing plants, leading to contamination of processed, ready-to-eat foods. L. monocytogenes persistence in various food processing environments (FPEs) has been extensively investigated by various subtyping tools, with increasing use of whole genome sequencing (WGS). However, major knowledge gaps remain. There is a need for facility-specific molecular signatures not only for adequate attribution of L. monocytogenes to a specific FPE but also for improved understanding of the ecology and evolution of L. monocytogenes in the food processing ecosystem. Furthermore, multiple strains can be recovered from a single FPE sample, but their diversity can be underestimated with common molecular subtyping tools. In this study we investigated a panel of 54 L. monocytogenes strains from four turkey processing plants in the United States. A combination of WGS and phenotypic assays was employed to assess strain persistence as well as identify facility-specific molecular signatures. Comparative analysis of allelic variation across the whole genome revealed that allelic profiles have the potential to be specific to individual processing plants. Certain allelic profiles remained associated with individual plants even when closely-related strains from other sources were included in the analysis. Furthermore, for certain sequence types (STs) based on the seven-locus multilocus sequence typing scheme, presence and location of premature stop codons in inlA, inlB length, prophage sequences, and the sequence content of a genomic hotspot could serve as plant-specific signatures. Interestingly, the analysis of different isolates from the same environmental sample revealed major differences not only in serotype and ST, but even in the sequence content of strains of the same ST. This study highlights the potential for WGS data to be deployed for identification of facility-specific signatures, thus facilitating the tracking of strain movement through the food chain. Furthermore, deployment of WGS for intra-sample strain analysis allows for a more complete environmental surveillance of L. monocytogenes in food processing facilities, reducing the risk of failing to detect strains that may be clinically relevant and potentially novel.

P-049: The spatial sub-clonal architecture in newly diagnosed myeloma patients revealed by whole genome and single-cell sequencing

<h3>Introduciton</h3> Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal accumulations of myeloma cells called focal lesions (FLs) have been shown to be the hotspots of spatial tumor heterogeneity, which is characterized by unique tumor sub-clones at different sites in the bone marrow (BM). However, little is known about the sub-clonal architecture in FLs, the mechanisms leading to site-unique mutations, and the link between sub-clones in FLs and treatment-resistance. <h3>Methods</h3> To fill this gap in knowledge, we used a comprehensive approach to characterize CD138-sorted tumor cells from FLs and random BM aspirates (RBMA) in 12 newly diagnosed MM patients, applying whole genome sequencing (WGS), single-cell(sc)-RNA-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing. Sc data was generated using the 10X Genomics platform. Pre-processing of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV were used for downstream analysis. WGS data was analyzed using inhouse pipelines. Mutations, copy-number-variations and mutational signatures were called using mpileup, ACESeq and mmsig. Neoantigen epitopes were predicted using NeoPredPipe. <h3>Results</h3> In the majority of patients (n=10/12) we found significant differences in the chromosomal and mutational profiles between FLs and paired RBMAs from the iliac crest. To identify the mechanisms underlying heterogenous mutations, we performed mutational signature analysis and found COSMIC signature SBS18 to be enriched in these mutations, suggesting that they were caused by reactive oxygen species. Interestingly several site-unique mutations were predicted as potential neoantigens. Driver gene mutations associated with relapse such as KRAS, CYLD, CDKN2C and TP53, were more often seen in FL sub-clones. Using a combination of WGS, sc-RNA and sc-ATAC-sequencing to characterize these sub-clones in more detail, we found increased regulatory accessibility and expression of genes associated with disease aggressiveness and drug resistance such as CXCR4 and members of the NFKB- and interferon pathways. The latter implies that FLs could play a significant role in the development of treatment resistance. Indeed, comparing sub-clones at baseline and after high-dose melphalan and autologous stem cell transplantation in one patient, we demonstrate expansion of a single tumor cell, which was closely related to the main sub-clone from the baseline FL. <h3>Conclusion</h3> In conclusion, our data provides novel insights into the mechanisms underlying site-unique mutations and the sub-clonal architecture at different sites in the BM. The combination of bulk and sc-techniques showed that FLs are enriched for sub-clones with genetic, transcriptional and regulatory markers characteristic for aggressive disease and can be the source of relapse in MM-patients. This implies that targeting FLs is essential for achieving a cure of MM.

DMD/BMD - GENETICS: EP.115 RNA-seq analysis for Dystrophinopathy

Dystrophinopathy, including Duchenne and Becker muscular dystrophies (DMD/BMD), is the most common hereditary neuromuscular disease caused by mutations in the DMD gene. It is known that the deletions/duplications spanning one or more exons are the cause of the disease in approximately 70% of patients while the small mutations involving one or at most several nucleotides are responsible for the remaining 30%. Nowadays, a combination of multiplex ligation-dependent probe amplification (MLPA) and targeted resequencing are widely used for the diagnosis of dystrophinopathy. Nevertheless, around 1% of patients still remain undiagnosed and seem to carry deep intronic mutations creating an extra exon or mutations produced by chromosomal rearrangement, leading to abnormal transcript. To decipher such mutations, we performed RNA-seq on 23 undiagnosed DMD/BMD cases in whom no variant was identified albeit dystrophin was deficient on muscle biopsy. We also analyzed 6 DMD/BMD cases who had a missense mutation in DMD gene as we suspected aberrant splicing may be caused by the mutation in these cases. By RNA-seq, abnormal splicing events were seen in 11 of 23 undiagnosed cases, in whom we further confirmed the presence of intronic mutations that led to pseudo exon production. The remaining 12 cases showed low expression of DMD transcript but without obvious abnormal splicing. In addition, in 2 of 6 cases with missense mutation, skipping of the exon harboring the mutation was detected. The variants were located the last or second to last exon in 2 cases. The remaining 4 cases showed normal expression of DMD transcript. Still 12 cases remained unsolved even by RNA-seq. Combination of whole genome sequencing and long-read sequencing will be performed for these cases. Not only RNA-seq analysis complements the routine genetic testing of dystrophinopathy but may help uncover the actual pathomechanisms of “missense” variants.