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Sébastien Rodrigue Arne C. Materna Sonia C. Timberlake Matthew C. Blackburn Rex R. Malmstrom Eric J. Alm Sallie W. Chisholm 《PloS one》2010,5(7)
Background
Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved.Methodology/Principal Findings
We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read.Conclusions/Significance
This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing. 相似文献2.
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Background
There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats.Methodology/Principal Findings
Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads.Conclusions
Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 相似文献4.
The total number of prokaryotic cells on earth has been estimated to be approximately 4–6 × 1030, with the majority of these being uncharacterized. This diversity represents a vast genetic bounty that may be exploited for the discovery of novel genes, entire metabolic pathways and potentially valuable end‐products thereof. Metagenomics constitutes the functional and sequence‐based analysis of the collective microbial genomes (microbiome) in a particular environment or environmental niche. Herein, we review the most recent sequence‐based metagenomic analyses of some of the most microbiologically diverse locations on earth; including soil, marine water and the insect and human gut. Such studies have helped to uncover several previously unknown facts; from the true microbial diversity of extreme environments to the actual extent of symbiosis that exists in the insect and human gut. In this respect, metagenomics has and will continue to play an essential part in the new and evolving area of microbial systems biology. 相似文献
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Jan Kieleczawa 《Journal of biomolecular techniques》2008,19(5):335-341
Anecdotal and not well-established evidence implies that there could be some effect of primer proximity in relation to a difficult region on read length and sequence quality. In this paper we sequenced many different categories of difficult regions where primers were located at various distances in relation to such regions and we found that there is only weak, if any, correlation between primer proximity and read length or sequence quality. The occasional improvements observed in some studies could be related instead to more optimal primers or better quality DNA. We suggest that instead of trying to design primers at varying distances to a difficult region, sequence finishers concentrate on applying modified chemistries appropriate to a given difficult region. 相似文献
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宏基因组学诞生于上世纪90年代,是指不经过微生物培养阶段,采用直接提取环境中总DNA的方法,对微生物基因总和进行研究的一门新学科.宏基因组技术的出现,使得人们对占微生物总体99%以上不可培养微生物的研究成为现实,微生物基因的可探测空间显著增大.总的来说,目前宏基因组技术的应用主要分为两个方面:一方面是筛选功能基因,开发具有所需功能的蛋白;另一方面是通过对宏基因组文库进行分析,探讨在各种环境下微生物间相互作用和微生物与周围环境间相互影响的规律,以便我们能更加客观、全面地认识微生物世界.在宏基因组技术的应用范围被不断扩展的同时,围绕着宏基因组文库的构建和筛选、测序和分析等方面的研究已成为宏基因组学发展的主要推动力,宏基因组技术的进步将不断提升其应用价值. 相似文献
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Metagenomics: advances in ecology and biotechnology 总被引:13,自引:0,他引:13
This review highlights the significant advances which have been made in prokaryotic ecology and biotechnology due to the application of metagenomic techniques. It is now possible to link processes to specific microorganisms in the environment, such as the detection of a new phototrophic process in marine bacteria, and to characterise the metabolic cooperation which takes place in mixed species biofilms. The range of prokaryote derived products available for biotechnology applications is increasing rapidly. The knowledge gained from analysis of biosynthetic pathways provides valuable information about enzymology and allows engineering of biocatalysts for specific processes. The expansion of metagenomic techniques to include alternative heterologous hosts for gene expression and the development of sophisticated assays which enable screening of thousands of clones offers the possibility to find out even more valuable information about the prokaryotic world. 相似文献
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Alban Mathieu Tom O. Delmont Timothy M. Vogel Patrick Robe Renaud Nalin Pascal Simonet 《PloS one》2013,8(6)
The human skin microbiome could provide another example, after the gut, of the strong positive or negative impact that human colonizing bacteria can have on health. Deciphering functional diversity and dynamics within human skin microbial communities is critical for understanding their involvement and for developing the appropriate substances for improving or correcting their action. We present a direct PCR-free high throughput sequencing approach to unravel the human skin microbiota specificities through metagenomic dataset analysis and inter-environmental comparison. The approach provided access to the functions carried out by dominant skin colonizing taxa, including Corynebacterium, Staphylococcus and Propionibacterium, revealing their specific capabilities to interact with and exploit compounds from the human skin. These functions, which clearly illustrate the unique life style of the skin microbial communities, stand as invaluable investigation targets for understanding and potentially modifying bacterial interactions with the human host with the objective of increasing health and well being. 相似文献
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Modern biotechnology has a steadily increasing demand for novel genes for application in various industrial processes and
development of genetically modified organisms. Identification, isolation and cloning for novel genes at a reasonable pace
is the main driving force behind the development of unprecedented experimental approaches. Metagenomics is one such novel
approach for engendering novel genes. Metagenomics of complex microbial communities (both cultivable and uncultivable) is
a rich source of novel genes for biotechnological purposes. The contributions made by metagenomics to the already existing
repository of prokaryotic genes is quite impressive but nevertheless, this technique is still in its infancy. In the present
review we have drawn comparison between routine cloning techniques and metagenomic approach for harvesting novel microbial
genes and described various methods to reach down to the specific genes in the metagenome. Accomplishments made thus far,
limitations and future prospects of this resourceful technique are discussed. 相似文献
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Metagenomics: DNA sequencing of environmental samples 总被引:2,自引:0,他引:2
Although genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent only a fraction of the living or once-living organisms of interest. Many species are difficult to study in isolation because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. Methods that are based on DNA sequencing circumvent these obstacles, as DNA can be isolated directly from living or dead cells in various contexts. Such methods have led to the emergence of a new field, which is referred to as metagenomics. 相似文献
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Vecherskii M. V. Semenov M. V. Lisenkova A. A. Stepankov A. A. 《Biology Bulletin》2022,48(3):S107-S117
Biology Bulletin - The prospects for application of metagenomic technologies in environmental studies are discussed. The advantages in investigating the taxonomic composition of aquatic and... 相似文献
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Different industries have different motivations to probe the enormous resource that is uncultivated microbial diversity. Currently, there is a global political drive to promote white (industrial) biotechnology as a central feature of the sustainable economic future of modern industrialized societies. This requires the development of novel enzymes, processes, products and applications. Metagenomics promises to provide new molecules with diverse functions, but ultimately, expression systems are required for any new enzymes and bioactive molecules to become an economic success. This review highlights industrial efforts and achievements in metagenomics. 相似文献