BACKGROUND: Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. METHODS: The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood-saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. RESULTS: The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood-saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030-0.9998 for SNVs and between 0.8883-0.9991 for small-indels in the case of the WGS protocol, and between 0.8643-0.999 for SNVs and between 0.7781-1.000 for small-indels in the case of the WES protocol. CONCLUSION: Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.
- MeSH
- DNA genetika MeSH
- exom MeSH
- genom lidský MeSH
- genomika MeSH
- lidé MeSH
- metagenomika * MeSH
- sekvenování celého genomu MeSH
- sekvenování exomu MeSH
- sliny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.
- MeSH
- centromera MeSH
- histonový kód MeSH
- homologní rekombinace * MeSH
- nukleozomy metabolismus MeSH
- proteiny buněčného cyklu antagonisté a inhibitory metabolismus MeSH
- regulace genové exprese u hub * MeSH
- represorové proteiny fyziologie MeSH
- Schizosaccharomyces pombe - proteiny antagonisté a inhibitory metabolismus fyziologie MeSH
- Schizosaccharomyces genetika MeSH
- transkripční faktory antagonisté a inhibitory metabolismus MeSH
- umlčování genů * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.
- MeSH
- biologické markery metabolismus MeSH
- deoxyribonukleasy metabolismus MeSH
- DNA krev metabolismus MeSH
- endoskopie MeSH
- extracelulární pasti účinky léků metabolismus MeSH
- extracelulární prostor metabolismus MeSH
- kolitida krev chemicky indukované patologie MeSH
- mitochondriální DNA krev MeSH
- myši inbrední C57BL MeSH
- ornithin analogy a deriváty farmakologie MeSH
- peptidylarginindeiminasa typu 4 metabolismus MeSH
- síran dextranu MeSH
- streptonigrin farmakologie MeSH
- střeva účinky léků patologie MeSH
- střevní sliznice účinky léků patologie MeSH
- stupeň závažnosti nemoci MeSH
- zánět krev patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. Increased stress tolerance, including the thermotolerance of some Cronobacter strains, can promote their survival in production facilities and thus raise the possibility of contamination of dried infant formula which has been identified as a potential source of infection. Some Cronobacter strains contain a genomic island, which might be responsible for increased thermotolerance. By analysis of Cronobacter sequenced genomes this determinant was found to be present in only 49/73 Cronobacter sakazakii strains and in 9/14 Cronobacter malonaticus strains. The island was also found in 16/17 clinical isolates originating from two hospitals. Two configurations of the locus were detected; the first one with the size of 18 kbp containing the thrB-Q genes and a shorter version (6 kbp) harbouring only the thrBCD and thrOP genes. Strains containing the thermotolerance island survived significantly better at 58 °C comparing to a C. sakazakii isogenic mutant lacking the island and strains with the longer version of the island were 2-10 times more tolerant than those with the shortened sequence. The function of the genomic island was further confirmed by its cloning into a low-copy vector and transforming it into the isogenic mutant. Different levels of rpoS, encoding for stress-response sigma factor, expression were also associated with variability in strain thermotolerance.
- MeSH
- bakteriální geny MeSH
- biologická adaptace genetika MeSH
- Cronobacter klasifikace genetika metabolismus MeSH
- enterobakteriální infekce mikrobiologie MeSH
- genom bakteriální * MeSH
- genomové ostrovy * MeSH
- infekce spojené se zdravotní péčí MeSH
- klonování DNA MeSH
- lidé MeSH
- multilokusová sekvenční typizace MeSH
- plazmidy genetika MeSH
- pořadí genů MeSH
- reakce na tepelný šok genetika MeSH
- teplota * MeSH
- testy genetické komplementace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.
- MeSH
- glukuronidasa genetika MeSH
- koně MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční homologie nukleových kyselin MeSH
- shluková analýza MeSH
- Streptococcus equi genetika MeSH
- terciární struktura proteinů genetika MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Úvod: Vplyv genetických faktorov na etiológiu nešpecifických črevných zápalov (IBD) je významnejší pri Crohnovej chorobe (CD) ako pri ulceróznej kolitíde (UC). Klinickí gastroenterológovia sa zamerali najmä na nosičstvo polymorfizmu génu NOD2/CARD15 a jeho fenotypovú manifestáciu vzhľadom na vek vzniku ochorenia, jeho lokalizáciu a priebeh. Ciele práce: 1. Zistiť prevalenciu polymorfizmov génu NOD2/CARD15 v súbore pacientov s CD a s UC v porovnaní s kontrolnou skupinou, 2. Potvrdiť vzťah medzi nosičstvom génového polymorfizmu NOD2/CARD15 a a) predpokladaným vznikom ochorenia v nižšom veku, b) so suponovanou častejšou lokalizáciou v terminálnom ileu ako aj c) s predominantne strikturizujúcou formou ochorenia. Materiál a metódy: Vyšetrovaný súbor tvorilo 52 pacientov s CD, 41 pacientov s UC a 22 kontrolných jedincov bez IBD. Jednotlivé nukleotidové polymorfizmy génu NOD2/CARD15 spojené s CD (R703W, G9O8R a 1007fs) boli zisťované metódou polymerázovej reťazovej reakcie s následným štiepením špecifickými reštrikčnými endonukleázami. Po stanovení alelovej frekvencie týchto polymorfizmov bola špecifikovaná genotypovo-fenotypová korelácia. Rozdiely vo výskyte alelových frekvencií medzi IBD pacientmi a kontrolami ako aj fenotypové rozdiely boli štatisticky analyzované pomocou ANOVA testu. Výsledky: Alelová frekvencia sledovaných génových polymorfizmov NOD2/CARD15 bola signifikantne vyššia v súbore CD pacientov (30/52) než u UC pacientov (8/41, p = 0,007) ako aj v porovnaní s kontrolnou skupinou (3/22, p = 0,003), čo platilo najmä pre polymorfizmus génu 1007fs (p = 0,004, resp. p = 0,023) a pre homozygótov (p = 0,072, p = 0,014). Analýza genotypovo-fenotypových korelácii dokázala, že polymorfizmus génu NOD2/ CARD15 bol štatisticky významne spojený s nižším vekom pri vzniku CD (do 16 rokov, p = 0,001), Asociácie s lokalizáciou v terminálnom ileu a so strikturizujúcou formou CD neboli potvrdené (p = 0,189 a p = 0,270). Záver: V predkladanej štúdii sme potvrdili na Slovensku doteraz nezisťovanú asociáciu variant génu NOD2/CARD15 s CD, ktorá sa fenotypovo manifestuje nižším vekom pri vzniku tohto ochorenia.
Introduction: Influence of genetic factors in the aethiology of inflammatory bowel disease (IBD) is more important in Crohn's disease (CD) than in ulcerative colitis (UC). Clinical gastroenterologists have focused on the consequences of being a NOD2/CARD15 gene polymorphism carrier with respect to onset, localisation and behaviour of CD. The aims of the study: 1. To investigate the prevalence of polymorphisms in NOD2/CARD15 gene in Slovak patients with CD and with UC in comparison with healthy controls, 2. confirm the link between NOD2/CARD15 gene mutations and a) the supposedly lower age at disease onset, b) localisation in terminal ileum, c) the predominatly stricturizing form of CD. Set of patients and methods: In total 93 IBD patients (52 CD and 41 UC) and 22 controls without IBD were genotyped. The single nucleotide polymorphisms (SNP) NOD2/CARD15 gene associated with CD (R702W, G908R and 1007fs) were assessed using the restriction fragments lenght polymorphisms polymerase chain reaction in IBD patients and in healthy controls. The allele frequency of these polymorphisms was determined and genotype-phenotype correlation was specified. The difference in allele frequencies between IBD patients and the controls as well as differences between phenotypes were analysed statistically using ANOVA test. Results: The allele frequencies of studied NOD2/ CARD15 SNP were significantly higher in CD (30/52) compared with UC patients (8/41, p = 0.007) and with healthy controls (3/22, p = 0.003). This applied for the frequency of 1007fs polymorphism in particular (p = 0.004 and p = 0.023 respectively for UC and healthy controls), especially for homozygotes (p = 0.072, p = 0.014). In the analysis of genotype-phenotype correlations, NOD2/CARD15 gene polymorphisms were significantly associated with early-onset CD (under 16 years, p = 0.001). The association with terminal ileum localisation and stricturizing form of disease was not found (p = 0.1989 and 0.270 irresp.). Conclusion: In the present study, we confirmed the association of NOD2/CARD15 variants with CD in a population not studied so far. In addition, this association is characterized by a gene dose effect and the studied NOD2/CARD15 variants are related to early-onset disease.
- MeSH
- Crohnova nemoc diagnóza etiologie genetika MeSH
- financování organizované MeSH
- lidé MeSH
- polymorfismus genetický genetika imunologie MeSH
- signální adaptorový protein Nod2 genetika imunologie MeSH
- statistika jako téma MeSH
- studie případů a kontrol MeSH
- ulcerózní kolitida diagnóza etiologie genetika MeSH
- věkové faktory MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Slovenská republika MeSH
