Forward-directed genetic screens are extremely powerful in identifying novel genes involved in a specific biological process, including various chromatin regulatory pathways. However, the traditional ways of genetic mapping are time- and cost-demanding. Recently, the whole process was revolutionized by the development of mapping-by-sequencing (MBS) protocols. In MBS, the causal mutations and their positions within genes are identified directly by whole-genome sequencing and bioinformatics analysis of the bulk of mutant plants selected based on the mutant phenotype from a segregating population. MBS increases precision and economizes the mapping. Here, we describe a general protocol and provide practical tips on how to proceed with the mapping-by-sequencing on the example of Arabidopsis forward-directed genetic screen designed to identify mutants sensitive to a specific type of DNA damage. The described protocol is generally applicable to a wide range of genetic screens in various inbreeding species with a reference genome sequence.

• Klíčová slova
• DNA damage repair, DNA-protein crosslinks, Forward genetics, Genetic mapping, High-throughput sequencing, Mapping-by-sequencing, SNP calling, Zebularine,
• MeSH
• Arabidopsis * genetika   MeSH
• fenotyp MeSH
• genom rostlinný MeSH
• mapování chromozomů * metody   MeSH
• mutace MeSH
• sekvenování celého genomu metody   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování * metody   MeSH
• Publikační typ
• časopisecké články MeSH

A double primary colorectal cancer (CRC) in a familial setting signals a high risk of CRC. In order to identify novel CRC susceptibility genes, we whole-exome sequenced germline DNA from nine persons with a double primary CRC and a family history of CRC. The detected variants were processed by bioinformatics filtering and prioritization, including STRING protein-protein interaction and pathway analysis. A total of 150 missense, 19 stop-gain, 22 frameshift and 13 canonical splice site variants fulfilled our filtering criteria. The STRING analysis identified 20 DNA repair/cell cycle proteins as the main cluster, related to genes CHEK2, EXO1, FAAP24, FANCI, MCPH1, POLL, PRC1, RECQL, RECQL5, RRM2, SHCBP1, SMC2, XRCC1, in addition to CDK18, ENDOV, ZW10 and the known mismatch repair genes. Another STRING network included extracellular matrix genes and TGFβ signaling genes. In the nine whole-exome sequenced patients, eight harbored at least two candidate DNA repair/cell cycle/TGFβ signaling gene variants. The number of families is too small to provide evidence for individual variants but, considering the known role of DNA repair/cell cycle genes in CRC, the clustering of multiple deleterious variants in the present families suggests that these, perhaps jointly, contributed to CRC development in these families.

• Klíčová slova
• familial colorectal cancer, germline variant, multiple primaries, whole‐exome sequencing,
• MeSH
• dospělí MeSH
• genetická predispozice k nemoci * MeSH
• kolorektální nádory * genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• oprava DNA genetika   MeSH
• rodokmen MeSH
• sekvenování exomu * metody   MeSH
• senioři MeSH
• zárodečné mutace * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Juxtaglomerular cell tumor (JxGCT) is a rare type of renal neoplasm demonstrating morphologic overlap with some mesenchymal tumors such as glomus tumor (GT) and solitary fibrous tumor (SFT). Its oncogenic drivers remain elusive, and only a few cases have been analyzed with modern molecular techniques. In prior studies, loss of chromosomes 9 and 11 appeared to be recurrent. Recently, whole-genome analysis identified alterations involving genes of MAPK-RAS pathway in a subset, but no major pathogenic alterations have been discovered in prior whole transcriptome analyses. Considering the limited understanding of the molecular features of JxGCTs, we sought to assess a collaborative series with a multiomic approach to further define the molecular characteristics of this entity. Fifteen tumors morphologically compatible with JxGCTs were evaluated using immunohistochemistry for renin, single-nucleotide polymorphism array (SNP), low-pass whole-genome sequencing, and RNA sequencing (fusion assay). In addition, methylation analysis comparing JxGCT, GT, and SFT was performed. All cases tested with renin (n=11) showed positive staining. Multiple chromosomal abnormalities were identified in all cases analyzed (n=8), with gains of chromosomes 1p, 10, 17, and 19 and losses of chromosomes 9, 11, and 21 being recurrent. A pathogenic HRAS mutation was identified in one case as part of the SNP array analysis. Thirteen tumors were analyzed by RNA sequencing, with 2 revealing in-frame gene fusions: TFG::GPR128 (interpreted as stochastic) and NAB2::STAT6 . The latter, originally diagnosed as JxGCT, was reclassified as SFT and excluded from the series. No fusions were detected in the remaining 11 cases; of note, no case harbored NOTCH fusions previously described in GT. Genomic methylation analysis showed that JxGCT, GT, and SFT form separate clusters, confirming that JxGCT represents a distinct entity (ie, different from GT). The results of our study show that JxGCTs are a distinct tumor type with a recurrent pattern of chromosomal imbalances that may play a role in oncogenesis, with MAPK-RAS pathway activation being likely a driver in a relatively small subset.

• MeSH
• dospělí MeSH
• epigeneze genetická MeSH
• epigenomika MeSH
• fúze genů * MeSH
• genetická predispozice k nemoci MeSH
• genomika MeSH
• imunohistochemie MeSH
• jednonukleotidový polymorfismus MeSH
• juxtaglomerulární aparát patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA MeSH
• nádorové biomarkery * genetika   MeSH
• nádory ledvin * genetika   patologie   chemie   MeSH
• sekvenování celého genomu MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Názvy látek
• nádorové biomarkery * MeSH

OBJECTIVES: While the reported incidence of non-tuberculous mycobacterial (NTM) infections is increasing, the true prevalence remains uncertain due to limitations in diagnostics and surveillance. The emergence of rare and novel species underscores the need for characterization to improve surveillance, detection, and management. METHODS: We performed whole-genome sequencing (WGS) and/or targeted deep-sequencing using the Deeplex Myc-TB assay on all NTM isolates collected in Slovakia and the Czech Republic between the years 2019 to 2023 that were unidentifiable at the species level by the routine diagnostic line probe assays (LPA) GenoType CM/AS and NTM-DR. Minimal inhibitory concentrations against amikacin, ciprofloxacin, moxifloxacin, clarithromycin, and linezolid were determined, and clinical data were collected. RESULTS: Twenty-eight cultures from different patients were included, of which 9 (32.1%) met the clinically relevant NTM disease criteria. The majority of those had pulmonary involvement, while two children presented with lymphadenitis. Antimycobacterial resistance rates were low. In total, 15 different NTM species were identified, predominantly rare NTM like M. neoaurum, M. kumamotonense and M. arupense. Notably, clinically relevant M. chimaera variants were also identified with WGS and Deeplex-Myc TB, which, unlike other M. chimaera strains, appeared to be undetectable by LPA assays. Deeplex detected four mixed infections that were missed by WGS analysis. In contrast, WGS identified two novel species, M. celatum and M. branderi, which were not detected by Deeplex-Myc TB. Importantly, one of these novel species strains was associated with clinically relevant pulmonary disease. DISCUSSION: Our study demonstrates the clinical relevance of uncommon NTM and the effectiveness of targeted deep-sequencing combined with WGS in identifying rare and novel NTM species.

• Klíčová slova
• Diagnostics, Non-tuberculous mycobacteria, Novel species, Targeted next-generation sequencing, Whole-genome sequencing,
• MeSH
• antibakteriální látky * farmakologie   MeSH
• atypické mykobakteriální infekce * mikrobiologie   diagnóza   MeSH
• bakteriální léková rezistence MeSH
• dítě MeSH
• dospělí MeSH
• genotyp MeSH
• klinická relevance MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikrobiální testy citlivosti * MeSH
• mladiství MeSH
• netuberkulózní mykobakterie * účinky léků   genetika   izolace a purifikace   MeSH
• předškolní dítě MeSH
• sekvenování celého genomu * MeSH
• senioři MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Slovenská republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky * MeSH

The opportunistic pathogen Candida parapsilosis is a major causative agent of candidiasis leading to death in immunocompromised individuals. Azoles are the first line of defense in their treatment. The purpose of this study was to characterize eight fluconazole-resistant and sensitive C. parapsilosis hospital isolates through a battery of phenotypic tests that target pathogenicity attributes such as virulence, biofilm formation, stress resistance, and ergosterol content. Whole genome sequencing was carried out to identify mutations in key pathogenicity and resistance genes. Phylogenetic comparison was performed to determine strain relatedness and clonality. Genomic data and phylogenetic analysis revealed that two isolates were C. orthopsilosis and C. metapsilosis misidentified as C. parapsilosis. Whole genome sequencing analysis revealed known and novel mutations in key drug resistance and pathogenicity genes such as ALS6, ALS7, SAPP3, SAP7, SAP9, CDR1, ERG6, ERG11 and UPC2. Phylogenetic analysis revealed a high degree of relatedness and clonality within our C. parapsilosis isolates. Our results showed that resistant isolates exhibited an increase in biofilm content compared to the sensitive isolates. In conclusion, our study is the first of its kind in Lebanon to describe phenotypic and genotypic characteristics of nosocomial C. parapsilosis complex isolates having a remarkable ability to form biofilms.

• Klíčová slova
• Candida parapsilosis, Biofilm, Clonality, Fluconazole, Pathogenicity, Resistance,
• MeSH
• antifungální látky * farmakologie   MeSH
• biofilmy * růst a vývoj   MeSH
• Candida parapsilosis * genetika   izolace a purifikace   klasifikace   MeSH
• fenotyp * MeSH
• flukonazol farmakologie   MeSH
• fungální léková rezistence * genetika   MeSH
• fylogeneze * MeSH
• genotyp * MeSH
• infekce spojené se zdravotní péčí mikrobiologie   MeSH
• kandidóza * mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti * MeSH
• nemocnice MeSH
• sekvenování celého genomu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Libanon MeSH
• Názvy látek
• antifungální látky * MeSH
• flukonazol MeSH

PURPOSE: Genetic testing in consanguineous families advances the general comprehension of pathophysiological pathways. However, short stature (SS) genetics remain unexplored in a defined consanguineous cohort. This study examines a unique pediatric cohort from Sulaimani, Iraq, aiming to inspire a genetic testing algorithm for similar populations. METHODS: Among 280 SS referrals from 2018-2020, 64 children met inclusion criteria (from consanguineous families; height ≤ -2.25 SD), 51 provided informed consent (30 females; 31 syndromic SS) and underwent investigation, primarily via exome sequencing. Prioritized variants were evaluated by the American College of Medical Genetics and Genomics standards. A comparative analysis was conducted by juxtaposing our findings against published gene panels for SS. RESULTS: A genetic cause of SS was elucidated in 31 of 51 (61%) participants. Pathogenic variants were found in genes involved in the GH-IGF-1 axis (GHR and SOX3), thyroid axis (TSHR), growth plate (CTSK, COL1A2, COL10A1, DYM, FN1, LTBP3, MMP13, NPR2, and SHOX), signal transduction (PTPN11), DNA/RNA replication (DNAJC21, GZF1, and LIG4), cytoskeletal structure (CCDC8, FLNA, and PCNT), transmembrane transport (SLC34A3 and SLC7A7), enzyme coding (CYP27B1, GALNS, and GNPTG), and ciliogenesis (CFAP410). Two additional participants had Silver-Russell syndrome and 1 had del22q.11.21. Syndromic SS was predictive in identifying a monogenic condition. Using a gene panel would yield positive results in only 10% to 33% of cases. CONCLUSION: A tailored testing strategy is essential to increase diagnostic yield in children with SS from consanguineous populations.

• Klíčová slova
• Consanguinity, Genetic testing algorithm, Pediatric endocrinology, Short stature, Short stature genes,
• MeSH
• algoritmy MeSH
• dítě MeSH
• genetické testování * metody   MeSH
• lidé MeSH
• mladiství MeSH
• mutace genetika   MeSH
• nanismus genetika   diagnóza   MeSH
• pokrevní příbuzenství * MeSH
• poruchy růstu genetika   diagnóza   MeSH
• předškolní dítě MeSH
• rodokmen MeSH
• sekvenování exomu metody   MeSH
• tělesná výška genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Irák MeSH

Halophilic bacteria are extremophiles that thrive in saline environment. Their ability to withstand such harsh conditions makes them an ideal choice for industrial applications such as lignocellulosic biomass degradation. In this study, a halophilic bacterium with the ability to produce extracellular cellulases and hemicellulases, designated as Nesterenkonia sp. CL21, was isolated from mangrove sediment in Tanjung Piai National Park, Malaysia. Thus far, studies on lignocellulolytic enzymes concerning bacterial species under this genus are limited. To gain a comprehensive understanding of its lignocellulose-degrading potential, the whole genome was sequenced using the Illumina NovaSeq 6000 platform. The genome of strain CL21 was assembled into 25 contigs with 3,744,449 bp and a 69.74% GC content and was predicted to contain 3,348 coding genes. Based on taxonomy analysis, strain CL21 shares 73.8 to 82.0% average nucleotide identity with its neighbouring species, below the 95% threshold, indicating its possible status as a distinct species in Nesterenkonia genus. Through in-depth genomic mining, a total of 81 carbohydrate-active enzymes were encoded. Among these, 24 encoded genes were identified to encompass diverse cellulases (GH3), xylanases (GH10, GH11, GH43, GH51, GH127 and CE4), mannanases (GH38 and GH106) and pectinases (PL1, PL9, and PL11). The production of lignocellulolytic enzymes was tested in the presence of several substrates. This study revealed that strain CL21 can produce a diverse array of enzymes which are active at different time points. By combining experimental data with genomic information, the ability of strain CL21 to produce lignocellulolytic enzymes has been elucidated, with potential applications in biorefinery industry.

• Klíčová slova
• Nesterenkonia, Genomics, Halophiles, Lignocellulolytic enzymes,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• celulasy genetika   metabolismus   MeSH
• fylogeneze * MeSH
• genom bakteriální * MeSH
• genomika * MeSH
• geologické sedimenty mikrobiologie   MeSH
• glykosidhydrolasy * genetika   metabolismus   MeSH
• lignin * metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenování celého genomu MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• celulasy MeSH
• glykosidhydrolasy * MeSH
• hemicellulase MeSH Prohlížeč
• lignin * MeSH
• lignocellulose MeSH Prohlížeč
• RNA ribozomální 16S MeSH

BACKGROUND: Treponema pallidum subspecies pertenue (TPE) is the causative agent of human and nonhuman primate (NHP) yaws infection. The discovery of yaws bacterium in wild populations of NHPs opened the question of transmission mechanisms within NHPs, and this work aims to take a closer look at the transmission of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our study determined eleven whole TPE genomes from NHP isolates collected from three national parks in Tanzania: Lake Manyara National Park (NP), Serengeti NP, and Ruaha NP. The bacteria were isolated from four species of NHPs: Chlorocebus pygerythrus (vervet monkey), Cercopithecus mitis (blue monkey), Papio anubis (olive baboon), and Papio cynocephalus (yellow baboon). Combined with previously generated genomes of TPE originating from NHPs in Tanzania (n = 11), 22 whole-genome TPE sequences have now been analyzed. Out of 231 possible combinations of genome-to-genome comparisons, five revealed an unexpectedly high degree of genetic similarity in samples collected from different NHP species, consistent with inter-species transmission of TPE among NHPs. We estimated a substitution rate of TPE of NHP origin, ranging between 1.77 × 10-7 and 3.43 × 10-7 per genomic site per year. CONCLUSIONS/SIGNIFICANCE: The model estimations predicted that the inter-species transmission happened recently, within decades, roughly in an order of magnitude shorter time compared to time needed for the natural diversification of all tested TPE of Tanzanian NHP origin. Moreover, the geographical separation of the sampling sites (NPs) does not preclude TPE transmission between and within NHP species.

• MeSH
• Cercopithecus aethiops MeSH
• Cercopithecus mikrobiologie   MeSH
• frambézie * mikrobiologie   přenos   MeSH
• fylogeneze * MeSH
• genom bakteriální MeSH
• lidé MeSH
• nemoci opic mikrobiologie   přenos   MeSH
• Papio anubis mikrobiologie   MeSH
• Papio cynocephalus mikrobiologie   genetika   MeSH
• primáti mikrobiologie   MeSH
• sekvenování celého genomu * MeSH
• Treponema pallidum genetika   izolace a purifikace   klasifikace   MeSH
• Treponema MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Tanzanie MeSH

BACKGROUND: The endosymbiotic relationship between Wolbachia bacteria and insects has been of interest for many years due to their diverse types of host reproductive phenotypic manipulation and potential role in the host's evolutionary history and population dynamics. Even though infection rates are high in Lepidoptera and specifically in butterflies, and reproductive manipulation is present in these taxa, less attention has been given to understanding how Wolbachia is acquired and maintained in their natural populations, across and within species having continental geographical distributions. RESULTS: We used whole genome sequencing data to investigate the phylogenetics, demographic history, and infection rate dynamics of Wolbachia in four species of the Spicauda genus of skipper butterflies (Lepidoptera: Hesperiidae), a taxon that presents sympatric and often syntopic distribution, with drastic variability in species abundance in the Neotropical region. We show that infection is maintained by high turnover rates driven mainly by pervasive horizontal transmissions, while also presenting novel cases of double infection by distantly related supergroups of Wolbachia in S. simplicius. CONCLUSIONS: Our results suggest that Wolbachia population dynamics is host species-specific, with genetic cohesiveness across wide geographical distributions. We demonstrate that low coverage whole genome sequencing data can be used for an exhaustive assessment of Wolbachia infection in natural populations of butterflies, as well as its dynamics in closely related host species. This ultimately leads to a better understanding of the endosymbiotic population dynamics of Wolbachia and its effects on the host's biology and evolution.

• Klíčová slova
• Wolbachia, Double infection, Hesperiidae, Historical demography, Phylogenetics, Population dynamics, Skipper butterflies,
• MeSH
• fylogeneze * MeSH
• genom bakteriální genetika   MeSH
• motýli * mikrobiologie   MeSH
• sekvenování celého genomu * MeSH
• symbióza * MeSH
• Wolbachia * genetika   klasifikace   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Taylorella equigenitalis is the causative agent of sexually transmitted contagious equine metritis. Infections manifest as cervicitis, vaginitis and endometritis and cause temporary infertility and miscarriages of mares. While previous studies have analyzed this organism for various parameters, the evolutionary dynamics of this pathogen, including the emergence of antibiotic resistance, remains unresolved. The aim of this study was to isolate contemporary strains, determine their genome sequences, evaluate their antibiotic resistance and compare them with other strains. We determined nine complete whole genome sequences of T. equigenitalis strains, mainly from samples collected from Kladruber horses in the Czech Republic. While T. equigenitalis strains from Kladruby isolated between 1982 and 2018 were inhibited by streptomycin, contemporary strains were found to be resistant to streptomycin, suggesting the recent emergence of this mutation. In addition, we used the collection dates of Kladruber horse strains to estimate the genome substitution rate, which resulted in a scaled mean evolutionary rate of 6.9×10-7 substitutions per site per year. Analysis with other available T. equigenitalis genome sequences (n = 18) revealed similarity of the Czech T. equigenitalis genomes with the Austrian T. equigenitalis genome, and molecular dating suggested a common ancestor of all analyzed T. equigenitalis strains from 1.5-2.6 thousand years ago, dating to the first centuries A.D. Our study revealed a recently emerged streptomycin resistance in T. equigenitalis strains from Kladruber horses, emphasizing the need for antibiotic surveillance and alternative treatments. Additionally, our findings provided insights into the pathogen's evolution rate, which is important for understanding its evolution and preparing preventive strategies.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• fylogeneze MeSH
• genom bakteriální * genetika   MeSH
• koně mikrobiologie   MeSH
• molekulární evoluce MeSH
• nemoci koní * mikrobiologie   MeSH
• sekvenování celého genomu * MeSH
• Taylorella equigenitalis * genetika   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• antibakteriální látky MeSH