Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
V současnosti nejsme schopni určit příčiny vzácných geneticky podmíněných nemocí u ~50% případů a počet nově objevovaných kauzálních genů v posledních letech klesá. Naším záměrem je provést integrovanou analýzu genomu, transkriptomu, proteomu a metabolomu k určení genetické diagnózy ve skupině ~40 případů a rodin se vzácným geneticky podmíněným onemocněním, u kterých předchozí cílená klinická, biochemická a genetická vyšetření včetně exomového sekvenování nevedly k diagnóze. Využijeme našich zkušeností s novými technologiemi celogenomového sekvenování (NovaSeq), sekvenování dlouhých fragmentů jednotlivých molekul DNA (Oxford Nanopore, PacBio) a možností korelací genomové informace s výsledky analýz tělních tekutin, tkání, tkáňových kultur a vhodných buněčných modelů. Uplatníme nové bioinformatické nástroje analýzy multi-OMIC dat a nástroje umožňující globální sdílení fenotypových a genomických dat. Cílem je zrychlit poznání doposud nediagnostikovaných vzácných nemocí , zlepšení diagnostické výtěžňosti a zajištění inovativní péče o pacienty s vzácnými nemocemi v České Republice.; The failure to diagnose the cause of a rare genetic disease occurs in ~50% of the cases and the rate of discovery of novel genes and disease-gene relations appears to be declining. We intend to apply multi-OMIC approaches to identify causal genetic defects in ~40 selected cases from our previous studies, in which we have negative results from standard genetic and genomic analyses. We will benefit from our access and experience with new platforms for whole-genome analysis (NovaSeq), single molecule long read length sequencing (Oxford Nanopore, PacBio) and our ability to correlate genomic information with transcriptome, proteome and metabolome analyses of affected tissues, body fluids and patient cell-derived models. We will also apply new bioinformatics tools allowing effective integration of OMIC data and use tools enabling exchange of phenotypic and genomic information via shared platforms and tools worldwide. The ultimate goal is to accelerate understanding of these unsolved diseases, improve diagnostic yield, and deliver innovative care for rare genetic diseases in Czech Republic.
Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
- MeSH
- Chromosomes, Plant genetics MeSH
- Cytosine * metabolism MeSH
- Epigenesis, Genetic MeSH
- Transcription, Genetic MeSH
- DNA Methylation * MeSH
- Gene Expression Regulation, Plant MeSH
- DNA, Ribosomal * genetics MeSH
- Gene Silencing * MeSH
- DNA Copy Number Variations MeSH
- Publication type
- Journal Article MeSH
Bovine genes TLR4 and TLR5, which encode antibacterial toll-like receptors, were screened for polymorphisms in Czech Red Pied (Czech Simmental) cattle to identify variants associated with reproduction, udder health, and milk production traits. Variants were discovered by hybrid resequencing of 164 bulls using HiSeq X-Ten and PacBio technologies and then individually genotyped. Nominal p-values < 0.05 for associations were detected in 18 combinations between 14 polymorphisms and 15 traits using one-way analysis of variance (ANOVA). The TLR4 variants g.610C>T (rs43578094) and g.10310T>G (rs8193072) in reference AC000135.1 were strictly associated with the index of early reproductive disorders and maternal calving ease, respectively, at false discovery rate (FDR) < 0.05. A highly permissive false discovery rate cutoff of 0.6 separated seventeen combinations in both genes comprising eight positives. In the case of the TLR4 variant g.9422T>C (rs8193060), indications were obtained for the association with as many as four reproductive traits: incidence of cystic ovaries, early reproductive disorders, calving ease, and production longevity. The permissive FDR interpretation for the TLR5 data indicated associations with cyst incidence and early reproduction disorders with maternal calving ease. Moreover, three TLR5 polymorphisms correlated with milk production traits. The discrepancy of the observed associations with the predicted impacts of the SNPs on protein function points to the role of haplotypes. Nevertheless, this question should be resolved on a larger scale. The observed associations are endorsed by independent evidence from the published functional roles in other species and by the published QTL mapping data.
- MeSH
- Polymorphism, Single Nucleotide MeSH
- Milk MeSH
- Immunity, Innate MeSH
- Reproduction genetics MeSH
- Cattle genetics MeSH
- Toll-Like Receptor 4 * genetics MeSH
- Toll-Like Receptor 5 * genetics MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Cattle genetics MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases. Because sequencing technologies use these enzymes, they might possess increased errors at non-B structures. To evaluate this, we analyzed error rates, read depth, and base quality of Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT) sequencing at non-B motifs. All technologies showed altered sequencing success for most non-B motif types, although this could be owing to several factors, including structure formation, biased GC content, and the presence of homopolymers. Single-nucleotide mismatch errors had low biases in HiFi and ONT for all non-B motif types but were increased for G-quadruplexes and Z-DNA in all three technologies. Deletion errors were increased for all non-B types but Z-DNA in Illumina and HiFi, as well as only for G-quadruplexes in ONT. Insertion errors for non-B motifs were highly, moderately, and slightly elevated in Illumina, HiFi, and ONT, respectively. Additionally, we developed a probabilistic approach to determine the number of false positives at non-B motifs depending on sample size and variant frequency, and applied it to publicly available data sets (1000 Genomes, Simons Genome Diversity Project, and gnomAD). We conclude that elevated sequencing errors at non-B DNA motifs should be considered in low-read-depth studies (single-cell, ancient DNA, and pooled-sample population sequencing) and in scoring rare variants. Combining technologies should maximize sequencing accuracy in future studies of non-B DNA.
Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.
- MeSH
- Genomics MeSH
- Giardia lamblia * genetics MeSH
- Giardia genetics MeSH
- Giardiasis * parasitology MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Helicobacter pylori (H. pylori) is a Gram-negative pathogen as a carcinogen of the class Ι, with unique genetic diversity and wide geographic differences. The high incidence of gastric cancer in East Asia may be related to the bacterial genotype. It is of great significance that the genome of H. pylori in East Asia is widely collected. Therefore, we combined two sequencing technologies (PacBio and Illumina HiSeq 4000) and multiple databases to sequence and annotate the whole genome of H. pylori GZ7 isolated from a gastric cancer patient in Guizhou, China. Furthermore, this sequence was further compared with the genome sequence of 23 H. pylori strains isolated from different regions through collinearity comparison, specific gene analysis, phylogenetic tree construction, etc. The results showed that the genome of H. pylori GZ7 consists of 1,579,995 bp circle chromosomes with a GC content of 39.51%. This chromosome has 1,572 coding sequences, three antibiotic resistance genes, five prophages, and 198 virulence genes. The comparative genome analyses showed that H. pylori GZ7 has 53 specific genes compared to the other 23 strains. Most of these specific genes have not been annotated and characterized until now, whose research may provide insights into the biological activities of this strain. H. pylori GZ7 has the closest genetic relationship with H. pylori F30, and the farthest genetic relationship with H. pylori ELS37, which indicates that H. pylori genomes have geographical differences. This information may provide a molecular basis and guidance for constructing diagnostic methods for H. pylori and researching subsequent experiments.
- MeSH
- Phylogeny MeSH
- Genome, Bacterial MeSH
- Helicobacter pylori * MeSH
- Helicobacter Infections * microbiology MeSH
- Humans MeSH
- Stomach Neoplasms * genetics microbiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
OBJECTIVES: The aim of the study is to characterise the genomic features of three GES-producing Enterobacterales isolates from Czech hospitals. METHODS: In 2020, during a routine screening of the hospital's surfaces in Prague General Hospital, two strains (CZ862 and CZ863) that belonged to the Enterobacter cloacae complex were found to be blaGES positive. Another blaGES positive strain identified as Klebsiella oxytoca was recovered from a patient hospitalised in Pilsen. Antibiotic susceptibility profiling was done with broth microdilution assay. Conjugation/transformation experiments were performed on all three strains. Genomic DNA of the three isolates was subjected to whole genome sequencing using PacBio platform. RESULTS: Multilocus sequence types typing of CZ862 and CZ863 identified the strains as ST837 and a novel ST (ST1622). Both blaGES harbouring plasmids showed high sequence similarity and complete query coverage (100% and 99.98%) with pEcl-35771cz. Both plasmids had two copies of blaGES instead of one copy as found in pEcl-35771cz. The clinical isolate CZ598 belonged to ST180. The plasmid harboured blaGES-7 gene, cat and aac(6')-lb and the novel variant blaOXA-1011. No similar sequences were observed, suggesting a novel plasmid. CONCLUSION: The detection of the two blaGES-positive plasmids in the same hospital environment, the first report after 3 years, suggests a hidden source. This highlights the importance of the hidden sources and evolution of such plasmids on the route of spreading into clinical settings. Also, the detection of the new blaOXA-1011, which is thought in this case to be associated with carbapenem resistance, imposes a health risk if disseminated, limiting therapeutic options.
- MeSH
- beta-Lactamases * genetics MeSH
- Genomics MeSH
- Klebsiella oxytoca * genetics MeSH
- Humans MeSH
- Plasmids genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Borrelia burgdorferi, the etiological agent of Lyme disease, persists in nature through an enzootic cycle consisting of a vertebrate host and an Ixodes tick vector. The sequence motifs modified by two well-characterized restriction/modification (R/M) loci of B. burgdorferi type strain B31 were recently described, but the methylation profiles of other Lyme disease Borrelia bacteria have not been characterized. Here, the methylomes of B. burgdorferi type strain B31 and 7 clonal derivatives, along with B. burgdorferi N40, B. burgdorferi 297, B. burgdorferi CA-11, B. afzelii PKo, B. afzelii BO23, and B. garinii PBr, were defined through PacBio single-molecule real-time (SMRT) sequencing. This analysis revealed 9 novel sequence motifs methylated by the plasmid-encoded restriction/modification enzymes of these Borrelia strains. Furthermore, while a previous analysis of B. burgdorferi B31 revealed an epigenetic impact of methylation on the global transcriptome, the current data contradict those findings; our analyses of wild-type B. burgdorferi B31 revealed no consistent differences in gene expression among isogenic derivatives lacking one or more restriction/modification enzymes. IMPORTANCE The principal causative agent of Lyme disease in humans in the United States is Borrelia burgdorferi, while B. burgdorferi, B. afzelii, and B. garinii, collectively members of the Borrelia burgdorferi sensu lato species complex, cause Lyme disease in Europe and Asia. Two plasmid-encoded restriction/modification systems have been shown to limit the genetic transformation of B. burgdorferi type strain B31 with foreign DNA, but little is known about the restriction/modification systems of other Lyme disease Borrelia bacteria. This paper describes the methylation motifs present on genomic DNAs of multiple B. burgdorferi, B. afzelii, and B. garinii strains. Contrary to a previous report, we did not find evidence for an epigenetic impact on gene expression by methylation. Knowledge of the motifs recognized and methylated by the restriction/modification enzymes of Lyme disease Borrelia will facilitate molecular genetic investigations of these important human pathogens. Additionally, the similar motifs methylated by orthologous restriction/modification systems of Lyme disease Borrelia bacteria and the presence of these motifs within recombinogenic loci suggest a biological role for these ubiquitous restriction/modification systems in horizontal gene transfer.
- MeSH
- Borrelia burgdorferi classification genetics MeSH
- DNA, Bacterial genetics MeSH
- Epigenomics * MeSH
- Humans MeSH
- Lyme Disease microbiology MeSH
- Methylation MeSH
- Nucleotide Motifs * MeSH
- Plasmids genetics metabolism MeSH
- Sequence Analysis, DNA * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, N.I.H., Intramural MeSH
Background: Invasive pneumococcal disease (IPD) remains a global health problem. IPD incidence has significantly decreased by the use of pneumococcal conjugate vaccines (PCV). Nevertheless, non-PCV serotypes remain a matter of concern. Eight Streptococcus pneumoniae serotype 24F isolates, belonging to a non-PCV serotype, were detected through the Lebanese Inter-Hospital Pneumococcal Surveillance Program. The aim of the study is to characterize phenotypic and genomic features of the 24F isolates in Lebanon. Methods: WGS using long reads sequencing (PacBio) was performed to produce complete circular genomes and to determine clonality, antimicrobial resistance and virulence determinants. Results: The sequencing results yielded eight closed circular genomes. Three multilocus sequence typing (MLST) types were identified (ST11618, ST14184, ST15253). Both MLST and WGS analyses revealed that these isolates from Lebanon were genetically homogenous belonging to clonal complex CC230 and clustered closely with isolates originating from Canada, United States of America, United Kingdom and Iceland. Their penicillin binding protein profiles correlated with both β-lactam susceptibility patterns and MLST types. Moreover, the isolates harbored the macrolide and tetracycline resistance genes and showed a similar virulence gene profile. To our knowledge, this study represents the first report of complete phenotypic and genomic characterization of the emerging Streptococcus pneumoniae, serotype 24F, in the Middle East and North Africa region.
- Publication type
- Journal Article MeSH
BACKGROUND: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. METHODS: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the blaVIM- and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). RESULTS: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the blaVIM-1 determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and blaVIM-1 on a non-conjugative IncA plasmid. CONCLUSIONS: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy.
- Publication type
- Journal Article MeSH
