mapping-by-sequencing
Dotaz
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2nd ed. x, 193 s., il.
sv.
- MeSH
- genomika MeSH
- mapování chromozomů MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- periodika MeSH
- Konspekt
- Obecná genetika. Obecná cytogenetika. Evoluce
- NLK Obory
- genetika, lékařská genetika
The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.
- MeSH
- biotechnologie metody MeSH
- chromozomy rostlin genetika MeSH
- genom rostlinný * MeSH
- mapování chromozomů metody MeSH
- pšenice genetika MeSH
- sekvenční analýza DNA metody MeSH
- tandemové repetitivní sekvence MeSH
- umělé bakteriální chromozomy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Physical maps created from large insert DNA libraries, typically cloned in BAC vector, are valuable resources for map-based cloning and de novo genome sequencing. The maps are most useful if contigs of overlapping DNA clones are anchored to chromosome(s), and ordered along them using molecular markers. Here we present a novel approach for anchoring physical maps, based on sequencing three-dimensional pools of BAC clones from minimum tilling path. RESULTS: We used physical map of wheat chromosome arm 3DS to validate the method with two different DNA sequence datasets. The first comprised 567 genes ordered along the chromosome arm based on syntenic relationship of wheat with the sequenced genomes of Brachypodium, rice and sorghum. The second dataset consisted of 7,136 SNP-containing sequences, which were mapped genetically in Aegilops tauschii, the donor of the wheat D genome. Mapping of sequence reads from individual BAC pools to the first and the second datasets enabled unambiguous anchoring 447 and 311 3DS-specific sequences, respectively, or 758 in total. CONCLUSIONS: We demonstrate the utility of the novel approach for BAC contig anchoring based on mass parallel sequencing of three-dimensional pools prepared from minimum tilling path of physical map. The existing genetic markers as well as any other DNA sequence could be mapped to BAC clones in a single in silico experiment. The approach reduces significantly the cost and time needed for anchoring and is applicable to any genomic project involving the construction of anchored physical map.
1 online zdroj
- MeSH
- mitochondriální DNA * MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- periodika MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- genetika, lékařská genetika
OBJECTIVE: To investigate whether mutations in the beta subunit of the epithelial sodium channel (Scnn1b) contribute to the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive rat. DESIGN: We determined the chromosome location of the rat Scnn1b gene, tested for cosegregation with blood pressure, and sequenced near full-length Scnn1b complementary DNAs (cDNAs) from SHR and Dahl salt-sensitive rats. METHODS: Chromosome mapping was performed by somatic cell hybrid analysis and by linkage analysis in recombinant inbred strains derived from SHR and Brown-Norway rats. Cosegregation analysis was performed by testing for correlations between blood pressure and Scnn1b genotypes in these strains. DNA sequencing was performed on cDNAs prepared from reverse-transcribed messenger RNA derived from rat kidney. RESULTS: The Scnn1b gene was closely linked to the Sa gene on rat chromosome 1. Blood pressure correlated significantly with Scnn1b gene in the recombinant inbred strains. Analysis of near full-length Scnn1b cDNAs from SHR and Dahl rats failed to reveal any coding sequence mutations that could affect the predicted amino acid sequence of the Scnn1b protein. CONCLUSION: The Scnn1b gene maps near the Sa gene in a region of rat chromosome 1 involved in the inherited control of blood pressure. If disordered activity of the epithelial cell sodium channel contributes to the pathogenesis of hypertension in the SHR or Dahl models, it must stem from genetic lesions in sequences that regulate Scnn1b function or in sequences important to the structure or function of the other sodium channel subunits.
- MeSH
- chlorid sodný farmakologie MeSH
- epitel metabolismus MeSH
- genetická vazba MeSH
- geny * MeSH
- hypertenze * genetika patofyziologie MeSH
- inbrední kmeny potkanů genetika MeSH
- krevní tlak MeSH
- krysa rodu rattus MeSH
- léková rezistence genetika MeSH
- mapování chromozomů * MeSH
- molekulární sekvence - údaje MeSH
- molekulární sondy genetika MeSH
- potkani inbrední SHR genetika MeSH
- rekombinace genetická MeSH
- sekvence nukleotidů MeSH
- sodíkové kanály * genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Methods in molecular biology ; vol. 255-256
1st ed. xx, 360 s.
Spot blotch (SB) caused by Bipolaris sorokiniana and powdery mildew (PM) caused by Blumeria graminis f. sp. hordei are two important diseases of barley. To map genetic loci controlling susceptibility and resistance to these diseases, a mapping population consisting of 138 recombinant inbred lines (RILs) was developed from the cross between Bowman and ND5883. A genetic map was constructed for the population with 852 unique single nucleotide polymorphism markers generated by sequencing-based genotyping. Bowman and ND5883 showed distinct infection responses at the seedling stage to two isolates (ND90Pr and ND85F) of Bipolaris sorokiniana and one isolate (Race I) of Blumeria graminis f. sp. hordei. Genetic analysis of the RILs revealed that one major gene (Scs6) controls susceptibility to Bipolaris sorokiniana isolate ND90Pr, and another major gene (Mla8) confers resistance to Blumeria graminis f. sp. hordei isolate Race I, respectively. Scs6 was mapped on chromosome 1H of Bowman, as previously reported. Mla8 was also mapped to the short arm of 1H, which was tightly linked but not allelic to the Rcs6/Scs6 locus. Quantitative trait locus (QTL) analysis identified two QTLs, QSbs-1H-P1 and QSbs-7H-P1, responsible for susceptibility to spot blotch caused by Bipolaris sorokiniana isolate ND85F in ND5883, which are located on chromosome 1H and 7H, respectively. QSbs-7H-P1 was mapped to the same region as Rcs5, whereas QSbs-1H-P1 may represent a novel allele conferring seedling stage susceptibility to isolate ND85F. Identification and molecular mapping of the loci for SB susceptibility and PM resistance will facilitate development of barley cultivars with resistance to the diseases.
Vzácná onemocnění představují skupinu 8000 různých nemocí postihujících zhruba 8 % populace. Studium genomů významně zvyšuje znalosti o genetické variabilitě člověka a pomocí efektivního sdílení dat v mezinárodních registrech umožňuje kauzální diagnostiku širokého spektra vzácných onemocnění u cca 55-65 % nemocných. Diagnostika zbývajících pacientů závisí na nových technologiích a konceptech studia lidských genomů, které se zaměřují na genetickou a funkční analýzu genetických variant a jejich kombinací s cílem rozpoznat genetickou heterogenitu jednotlivých nemocí, přítomnost somatického mozaicismu, existenci fenokopií, různé penetrance a expresivity jednotlivých mutací a oligogenních typů dědičnosti. Stoupá význam analýz nekódujících oblastí lidské DNA a studium jejího vlivu na transkripci a strukturu mRNA a analýzy repetitivních a homologních oblastí lidského genomu a interpretace jejich variability. Při hledání genetické příčiny vzácných nemocí je třeba cíleně analyzovat biologické tekutiny, tkáně i vhodné buněčné a zvířecí modely připravované metodami buněčného reprogramování nebo cílených změn genomu. Přinášíme stručný přehled metod, které zahrnují celoexomové a celogenomové sekvenování nové generace, funkční a homologní klonování, funkční komplementaci, mapování genů pomocí vazebné analýzy a porovnávání genomové informace jedince nebo skupiny jedinců s genetickou variabilitou populace. Exomovou analýzu jsme provedli u více než 520 pacientů s diagnostickou úspěšnosti nad 50 %. Srovnáním vlastních výsledků celoexomového sekvenování s výsledky cíleného sekvenování jsme v souboru 225 nemocných došli k závěru, že diagnosticky i ekonomicky je smysluplnější indikovat celoexomové a v blízké budoucnosti celogenomové sekvenování. Moderně koncipovaná diagnostika a výzkum vzácných nemocí je časově, personálně i finančně náročná a vyžaduje spolupráci lékařů s pracovišti biologicky orientovaného výzkumu. Jejich nezbytnost pro české zdravotnictví představuje výzvu pro organizátory i plátce českého zdravotnictví a výzkumu. Klíčová slova: vzácná a komplexní onemocnění, celoexomové sekvenování, mapování genů
Rare diseases represent a heterogeneous group of approximately 8000 various disorders and affect nearly 8 % of the population. The local and international studies of human genomes help to increase the knowledge about genetic variability of the man and due to effective sharing of clinical and molecular data in the registries enable casual diagnostics of the broad spectrum of rare and complex diseases in 55–65 % of the cases. With the diagnostics in the remaining group of patients, new methods and technologies studying human genome are of importance including genetic and functional analyses of genomic variants and their combinations with the aims to recognize and interpret the significances of the somatic mosaics, genetic heterogeneity of individual disorders, the presence of eventual phenocopy, different penetrance and expressivity of individual mutation and diseases with the oligogenic inheritance. Recently, the increasing significance of analyses of noncoding regions in human DNA were recognized including the impact of repetitive and homologs regions on transcription and structure of mRNA. For the diagnostics of genetic causality in patients is necessary to focus on analyses of biologic fluids, tissues, cultivated cells and animal models prepared by methods of cell reprogramming or directed mutagenesis. In this paper, the overview of methods and their importance and limitation is described including whole exome sequencing (WES), whole genome sequencing, functional and homolog cloning, functional complementation, mapping of genes with the help of binding analyses and matching of the results from individual genome with genetic variability in the adequate population. In our institutions, we performed WES in > 520 patients with successful diagnostics above 50 %. In addition, in our group of 225 patients with rare diseases we compared the result of WES with the results of direct sequencing of individual genes indicated by clinical geneticist from various regions of the country and we recognized much higher diagnostic and economic value of WES. Modern diagnostics of rare diseases is time and money consuming and requires close cooperation between patients, their families, attending physicians, clinical geneticists and experts from various laboratories involved in biologic oriented research. It represents a big challenge for organisers and payers of the health care system. Keywords: are diseases, complex disorders, whole exome sequencing, gene mapping
