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Fabian Ripp Christopher Felix Krombholz Yongchao Liu Mathias Weber Anne Sch?fer Bertil Schmidt Rene K?ppel Thomas Hankeln 《BMC genomics》2014,15(1)
Background
DNA-based methods like PCR efficiently identify and quantify the taxon composition of complex biological materials, but are limited to detecting species targeted by the choice of the primer assay. We show here how untargeted deep sequencing of foodstuff total genomic DNA, followed by bioinformatic analysis of sequence reads, facilitates highly accurate identification of species from all kingdoms of life, at the same time enabling quantitative measurement of the main ingredients and detection of unanticipated food components.Results
Sequence data simulation and real-case Illumina sequencing of DNA from reference sausages composed of mammalian (pig, cow, horse, sheep) and avian (chicken, turkey) species are able to quantify material correctly at the 1% discrimination level via a read counting approach. An additional metagenomic step facilitates identification of traces from animal, plant and microbial DNA including unexpected species, which is prospectively important for the detection of allergens and pathogens.Conclusions
Our data suggest that deep sequencing of total genomic DNA from samples of heterogeneous taxon composition promises to be a valuable screening tool for reference species identification and quantification in biosurveillance applications like food testing, potentially alleviating some of the problems in taxon representation and quantification associated with targeted PCR-based approaches.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-639) contains supplementary material, which is available to authorized users. 相似文献3.
The network theoretical framework of ecological community studies is expected to promote not only the basic understanding of ecological and coevolutionary dynamics but also the application of those scientific insights into ecosystem management. However, our knowledge of ecological network architecture in the wild largely stems from empirical studies on macro-organismal systems such as those of plant–pollinator, plant–seed disperser, and prey–predator interactions. In this sense, we have remained ignorant of the diversity of ecological network architecture, its underlying assembly processes, and its consequences on ecological and coevolutionary dynamics. In this paper, I discuss how the high-throughput DNA barcoding of microbes, especially that based on next-generation sequencing, potentially expands the target of ecological network studies. I review the methodological platforms of next-generation sequencing-based analyses of microbe–host animal/plant networks and then introduce some case studies on the networks of plants and their hyper-diverse fungal symbionts. As those preliminary studies are uncovering the unexpected diversity of ecological network architecture, further application of such next-generation sequencing-based analyses to a diverse array of microbial systems will significantly improve our views on community ecological and coevolutionary processes. 相似文献
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The Chinese walnut (Juglans cathayensis L.), valued for both its nut and wood, is an ecologically important tree species endemic temperate southern China. Investigation of the genetic diversity of Chinese walnut has been limited to natural population genetics and genetic germplasm resources. Here, we describe the development of 12 polymorphic microsatellite markers using next-generation sequencing to screen 96 Chinese walnut individuals collected from 11 natural populations. The number of alleles per locus ranged from 5 to 12. The observed heterozygosity (0.288–0.748) overlapped well with the expected heterozygosity (0.337–0.751). This species has high genetic diversity and gene flow among different populations (FST = 0.075, Nm = 3.088). These markers will be useful for future studies on population genetic structure, evolutionary ecology, and genetic breeding of this walnut tree or other Juglans species. 相似文献
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新一代测序技术(Next-generation sequencing,NGS)在阐明复杂和高度重复的基因组结构,DNA序列与基因组结构变异同重要农艺性状之间的关系等方面具有重要作用。从NGS系统的开发与作物基因组测序,NGS与转录组分析,NGS与全基因组关联图谱,及SNPs开发与预测育种等方面,综述了NGS技术在作物基因组研究中的应用,可为作物基因组研究提供理论基础。 相似文献
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Masu salmon, Oncorhynchus masou masou, is an economically important fish species in the Far East and occurs in two life history forms: sea-run migratory (anadromous) and freshwater resident (non-anadromous). The non-anadromous form has recently become a popular freshwater food and game fish during a well-known Korean winter festival. However, the genetic background of this species remains largely unknown, partly due to a lack of molecular genetic markers. In this study, we developed new polymorphic microsatellite markers for masu salmon using next-generation sequencing technology. From 40 primer sets, 11 primer sets (27.5% of the primer sets selected) were successfully amplified with 106 alleles (range 2–9) in 64 individuals from different populations: two wild and one hatchery. Observed and expected heterozygosities ranged from 0.304 to 0.947 and 0.278 to 0.865, respectively. Significant departures from the Hardy–Weinberg equilibrium were detected for four markers (OMM11, OMM17, OMM28, and OMM33) in a single population. All pair-wise FST values were highly significant between the wild and hatchery populations (range 0.084–0.183, P < 0.0001). We identified a set of robust microsatellite markers that worked well even in formalin-fixed samples, which will be suitable for biogeographical and population structure analyses of the masu salmon. 相似文献
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Bulbuls (family Pycnonotidae) are a diverse family of songbirds that carry out a number of ecologically important functions associated with seed dispersal. Since, 2003, a puff-throated bulbul (Alophoixus pallidus) population in the Mo-Singto Long-term Biodiversity Research Plot in Khao Yai National Park, Thailand has served as a model system for examining how bulbul behavior, movement, and demographics affect Southeast Asian forests. In this study, we used 454 pyrosequencing to discover microsatellites from A. pallidus that will enable the long-term mark-recapture work conducted at Mo-Singto to be complemented by molecular ecology and population genetic studies. In addition, we conducted fragment analysis to examine the level of genetic diversity exhibited by the Mo-Singto population. In total, we identified 103 DNA fragments containing microsatellite repeats and 66 fragments with sufficient flanking sequences to allow for primer design. Upon screening 26 loci via PCR-based genotyping assays, we identified nine polymorphic loci and used eight of these to examine genetic diversity in the Mo-Singto population. The results of these analyses suggest that the Mo-Singto population is moderately diverse (mean number of effective alleles across eight loci = 3.36, standard deviation = 1.78), is more-or-less in Hardy–Weinberg equilibrium, and has not recently been subject to severe population reduction. 相似文献
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Mathieu Almeida Agnès Hébert Anne-Laure Abraham Simon Rasmussen Christophe Monnet Nicolas Pons Céline Delbès Valentin Loux Jean-Michel Batto Pierre Leonard Sean Kennedy Stanislas Dusko Ehrlich Mihai Pop Marie-Christine Montel Fran?oise Irlinger Pierre Renault 《BMC genomics》2014,15(1)
Background
Microbial communities of traditional cheeses are complex and insufficiently characterized. The origin, safety and functional role in cheese making of these microbial communities are still not well understood. Metagenomic analysis of these communities by high throughput shotgun sequencing is a promising approach to characterize their genomic and functional profiles. Such analyses, however, critically depend on the availability of appropriate reference genome databases against which the sequencing reads can be aligned.Results
We built a reference genome catalog suitable for short read metagenomic analysis using a low-cost sequencing strategy. We selected 142 bacteria isolated from dairy products belonging to 137 different species and 67 genera, and succeeded to reconstruct the draft genome of 117 of them at a standard or high quality level, including isolates from the genera Kluyvera, Luteococcus and Marinilactibacillus, still missing from public database. To demonstrate the potential of this catalog, we analysed the microbial composition of the surface of two smear cheeses and one blue-veined cheese, and showed that a significant part of the microbiota of these traditional cheeses was composed of microorganisms newly sequenced in our study.Conclusions
Our study provides data, which combined with publicly available genome references, represents the most expansive catalog to date of cheese-associated bacteria. Using this extended dairy catalog, we revealed the presence in traditional cheese of dominant microorganisms not deliberately inoculated, mainly Gram-negative genera such as Pseudoalteromonas haloplanktis or Psychrobacter immobilis, that may contribute to the characteristics of cheese produced through traditional methods.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1101) contains supplementary material, which is available to authorized users. 相似文献10.
One of the major questions in microbial ecology is “who is there?” This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution. 相似文献
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Background
Human leukocyte antigen (HLA) is a group of genes that are extremely polymorphic among individuals and populations and have been associated with more than 100 different diseases and adverse drug effects. HLA typing is accordingly an important tool in clinical application, medical research, and population genetics. We have previously developed a phase-defined HLA gene sequencing method using MiSeq sequencing.Results
Here we report a simple, high-throughput, and cost-effective sequencing method that includes normalized library preparation and adjustment of DNA molar concentration. We applied long-range PCR to amplify HLA-B for 96 samples followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. After sequencing, we observed low variation in read percentages (0.2% to 1.55%) among the 96 demultiplexed samples. On this basis, all the samples were amenable to haplotype phasing using our phase-defined sequencing method. In our study, a sequencing depth of 800x was necessary and sufficient to achieve full phasing of HLA-B alleles with reliable assignment of the allelic sequence to the 8 digit level.Conclusions
Our HLA sequencing method optimized for 96 multiplexing samples is highly time effective and cost effective and is especially suitable for automated multi-sample library preparation and sequencing.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-645) contains supplementary material, which is available to authorized users. 相似文献12.
《Fungal Ecology》2022
The factors shaping the composition of microbial communities in trees remain poorly understood. We evaluated whether the core and satellite fungal communities in five pine species (Pinus radiata, Pinus pinaster, Pinus sylvestris, Pinus nigra, and Pinus uncinata) were shaped by the host species identity. Because the trees had earlier been inoculated with a fungal pathogen (Fusarium circinatum), we also explored the possibilities to detect its presence and potential co-occurrence networks. We found interspecific variation in the fungal community composition and abundance among the different tree species and the existence of a core microbiome that was independent of the host species. The presence of F. circinatum was confirmed in some samples through qPCR but the pathogen did not co-occur with a specific fungal community. The results highlight the importance of host species as a determinant of microbiome assembly in common environments. 相似文献
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Yuqing Yang Xin Wang Kaikun Xie Congmin Zhu Ning Chen Ting Chen 《基因组蛋白质组与生物信息学报(英文版)》2021,19(5):834-847
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This article reviews basic concepts,general applications,and the potential impact of next-generation sequencing(NGS)technologies on genomics,with particular reference to currently available and possible future platforms and bioinformatics.NGS technologies have demonstrated the capacity to sequence DNA at unprecedented speed,thereby enabling previously unimaginable scientific achievements and novel biological applications.But,the massive data produced by NGS also presents a significant challenge for data storage,analyses,and management solutions.Advanced bioinformatic tools are essential for the successful application of NGS technology.As evidenced throughout this review,NGS technologies will have a striking impact on genomic research and the entire biological field.With its ability to tackle the unsolved challenges unconquered by previous genomic technologies,NGS is likely to unravel the complexity of the human genome in terms of genetic variations,some of which may be confined to susceptible loci for some common human conditions.The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come. 相似文献
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João Carvalho Hernán E. Morales Rui Faria Roger K. Butlin Vítor C. Sousa 《Molecular ecology resources》2023,23(7):1737-1755
Next-generation sequencing of pooled samples (Pool-seq) is a popular method to assess genome-wide diversity patterns in natural and experimental populations. However, Pool-seq is associated with specific sources of noise, such as unequal individual contributions. Consequently, using Pool-seq for the reconstruction of evolutionary history has remained underexplored. Here we describe a novel Approximate Bayesian Computation (ABC) method to infer demographic history, explicitly modelling Pool-seq sources of error. By jointly modelling Pool-seq data, demographic history and the effects of selection due to barrier loci, we obtain estimates of demographic history parameters accounting for technical errors associated with Pool-seq. Our ABC approach is computationally efficient as it relies on simulating subsets of loci (rather than the whole-genome) and on using relative summary statistics and relative model parameters. Our simulation study results indicate Pool-seq data allows distinction between general scenarios of ecotype formation (single versus parallel origin) and to infer relevant demographic parameters (e.g. effective sizes and split times). We exemplify the application of our method to Pool-seq data from the rocky-shore gastropod Littorina saxatilis, sampled on a narrow geographical scale at two Swedish locations where two ecotypes (Wave and Crab) are found. Our model choice and parameter estimates show that ecotypes formed before colonization of the two locations (i.e. single origin) and are maintained despite gene flow. These results indicate that demographic modelling and inference can be successful based on pool-sequencing using ABC, contributing to the development of suitable null models that allow for a better understanding of the genetic basis of divergent adaptation. 相似文献
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Background
Second-generation sequencers generate millions of relatively short, but error-prone, reads. These errors make sequence assembly and other downstream projects more challenging. Correcting these errors improves the quality of assemblies and projects which benefit from error-free reads.Results
We have developed a general-purpose error corrector that corrects errors introduced by Illumina, Ion Torrent, and Roche 454 sequencing technologies and can be applied to single- or mixed-genome data. In addition to correcting substitution errors, we locate and correct insertion, deletion, and homopolymer errors while remaining sensitive to low coverage areas of sequencing projects. Using published data sets, we correct 94% of Illumina MiSeq errors, 88% of Ion Torrent PGM errors, 85% of Roche 454 GS Junior errors. Introduced errors are 20 to 70 times more rare than successfully corrected errors. Furthermore, we show that the quality of assemblies improves when reads are corrected by our software.Conclusions
Pollux is highly effective at correcting errors across platforms, and is consistently able to perform as well or better than currently available error correction software. Pollux provides general-purpose error correction and may be used in applications with or without assembly. 相似文献17.
The Leishmania homologue of activated C kinase (LACK) a known T cell epitope from soluble Leishmania antigens (SLA) that
confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains.
LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was
completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the
LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of
visceral Leishmaniasis vaccine target. 相似文献
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Adonney Allan de Oliveira Veras Pablo Henrique Caracciolo Gomes de Sá Vasco Azevedo Artur Silva Rommel Thiago Jucá Ramos 《Bioinformation》2013,9(16):840-841
Next-generation sequencing technologies have increased the amount of biological data generated. Thus, bioinformatics has become
important because new methods and algorithms are necessary to manipulate and process such data. However, certain challenges
have emerged, such as genome assembly using short reads and high-throughput platforms. In this context, several algorithms have
been developed, such as Velvet, Abyss, Euler-SR, Mira, Edna, Maq, SHRiMP, Newbler, ALLPATHS, Bowtie and BWA. However,
most such assemblers do not have a graphical interface, which makes their use difficult for users without computing experience
given the complexity of the assembler syntax. Thus, to make the operation of such assemblers accessible to users without a
computing background, we developed AutoAssemblyD, which is a graphical tool for genome assembly submission and remote
management by multiple assemblers through XML templates.
Availability
AssemblyD is freely available at https://sourceforge.net/projects/autoassemblyd. It requires Sun jdk 6 or higher. 相似文献20.
呼吸系统感染发病率高,早期明确感染的病原体是提高治愈率、降低死亡率的关键.目前病原体培养仍是临床病原学诊断的主要方式,但其敏感性低、耗时较长,不利于早期诊断和治疗.宏基因组学测序技术具有覆盖病原体广泛、快速、无偏倚、无需特异性扩增的优势,在鉴定罕见、混合感染、免疫抑制患者感染和常规检测方法难以检测到病原体的诊断中有较高... 相似文献