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BACKGROUND: Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. RESULTS: In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. CONCLUSION: A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection.
BACKGROUND: The investigation of plant genome structure and evolution requires comprehensive characterization of repetitive sequences that make up the majority of higher plant nuclear DNA. Since genome-wide characterization of repetitive elements is complicated by their high abundance and diversity, novel approaches based on massively-parallel sequencing are being adapted to facilitate the analysis. It has recently been demonstrated that the low-pass genome sequencing provided by a single 454 sequencing reaction is sufficient to capture information about all major repeat families, thus providing the opportunity for efficient repeat investigation in a wide range of species. However, the development of appropriate data mining tools is required in order to fully utilize this sequencing data for repeat characterization. RESULTS: We adapted a graph-based approach for similarity-based partitioning of whole genome 454 sequence reads in order to build clusters made of the reads derived from individual repeat families. The information about cluster sizes was utilized for assessing the proportion and composition of repeats in the genomes of two model species, Pisum sativum and Glycine max, differing in genome size and 454 sequencing coverage. Moreover, statistical analysis and visual inspection of the topology of the cluster graphs using a newly developed program tool, SeqGrapheR, were shown to be helpful in distinguishing basic types of repeats and investigating sequence variability within repeat families. CONCLUSIONS: Repetitive regions of plant genomes can be efficiently characterized by the presented graph-based analysis and the graph representation of repeats can be further used to assess the variability and evolutionary divergence of repeat families, discover and characterize novel elements, and aid in subsequent assembly of their consensus sequences.
Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.
- MeSH
- elektroforéza kapilární metody MeSH
- fylogeneze MeSH
- genotyp MeSH
- geny MHC třídy II MeSH
- molekulární sekvence - údaje MeSH
- Passeriformes klasifikace genetika MeSH
- polymorfismus konformace jednovláknové DNA MeSH
- ptačí proteiny genetika MeSH
- sekvenční analýza DNA metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.
- MeSH
- Daphnia parazitologie MeSH
- genetická variace * MeSH
- Mesomycetozoea klasifikace genetika MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- sekvenční analýza DNA * MeSH
- software MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Brown mud, as a waste product of the industrial process of aluminum production, represents a great environmental burden due to its toxicity to living organisms. However, some microorganisms are able to survive in this habitat, and they can be used in bioremediation processes. Traditional cultivation methods have a limited capacity to characterize bacterial composition in environmental samples. Recently, next-generation sequencing methods have provided new perspectives on microbial community studies. The aim of this study was to analyze the bacterial community in the drainage water of brown mud disposal site near Žiar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing. We obtained 9964 sequences assigned to 163 operational taxonomic units belonging to 10 bacterial phyla. The phylum Proteobacteria showed the highest abundance (80.39%) within the bacterial community, followed by Firmicutes (13.05%) and Bacteroidetes (5.64%). Other bacterial phyla showed an abundance lower than 1%. The classification yielded 85 genera. Sulfurospirillum spp. (45.19%) dominated the bacterial population, followed by Pseudomonas spp. (13.76%) and Exiguobacterium spp. (13.02%). These results indicate that high heavy metals content, high pH, and lack of essential nutrients are the drivers of a dramatic reduction of diversity in the bacterial population in this environment.
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- Bacteria klasifikace genetika izolace a purifikace MeSH
- biodegradace MeSH
- biodiverzita * MeSH
- chemické látky znečišťující vodu analýza metabolismus MeSH
- fylogeneze MeSH
- fyziologie bakterií * MeSH
- hutnictví MeSH
- koncentrace vodíkových iontů MeSH
- mikrobiální společenstva genetika MeSH
- odpadní voda chemie mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- těžké kovy analýza metabolismus MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Slovenská republika MeSH
Pseudomonas prosekii is a recently described species isolated exclusively from James Ross Island close to the Antarctic Peninsula at 64° south latitude. Here, we present two P. prosekii genome sequences and their analyses with respect to phylogeny, low temperature adaptation, and potential biotechnological applications. The genome of P. prosekii P2406 comprised 5,896,482 bp and 5324 genes (GC content of 59.71%); the genome of P. prosekii P2673 consisted of 6,087,670 bp and 5511 genes (GC content of 59.50%). Whole genome sequence comparisons confirmed a close relationship between both investigated strains and strain P. prosekii LMG 26867T. Gene mining revealed the presence of genes involved in stress response, genes encoding cold shock proteins, oxidative stress proteins, osmoregulation proteins, genes for the synthesis of protection molecules, and siderophores. Comparative genome analysis of P. prosekii and P. aeruginosa PAO1 highlighted differences in genome content between extremophile species and a mesophilic opportunistic pathogen.
- MeSH
- aklimatizace MeSH
- bakteriální proteiny genetika MeSH
- fylogeneze MeSH
- fyziologická adaptace MeSH
- genom bakteriální * MeSH
- mapování chromozomů MeSH
- nadmořská výška MeSH
- Pseudomonas genetika izolace a purifikace fyziologie MeSH
- sekvence nukleotidů MeSH
- sekvenování celého genomu MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Antarktida MeSH
OBJECTIVES: To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. METHODS: A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was performed using the 454-Genome Sequencer FLX system. The sequences were compared using bioinformatic tools with other sequenced IncHI1 plasmids. RESULTS: A comparative analysis of pEQ1 and pEQ2 identified high nucleotide identity with the IncHI1 type 2 plasmids. A novel 24 kb module containing an operon involved in short-chain fructooligosaccharide uptake and metabolism was found in the pEQ backbones. The role of the pEQ plasmids in the metabolism of short-chain fructooligosaccharides was demonstrated by studying the growth of E. coli cells in the presence of these sugars. The module containing the blaCTX-M-1 gene was formed by a truncated macrolide resistance cluster and flanked by IS26 as previously observed in IncI1 and IncN plasmids. The IncHI1 plasmid changed size and gained the quinolone resistance gene qnrS1 as a result of IS26-mediated fusion with an IncX1 plasmid. CONCLUSIONS: Our data highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses.
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- beta-laktamasy genetika MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- infekce vyvolané Escherichia coli mikrobiologie veterinární MeSH
- koně MeSH
- metabolické sítě a dráhy genetika MeSH
- molekulární evoluce * MeSH
- molekulární sekvence - údaje MeSH
- plazmidy * MeSH
- pořadí genů MeSH
- proteiny z Escherichia coli genetika MeSH
- sekvenční analýza DNA * MeSH
- syntenie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
