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1.
DNA sequences can be used for the analysis of genetic variation and gene function. The high-throughput sequencing techniques that have been developed over the past three years can read as many as one billion bases per run, and are far less expensive than the traditional Sanger sequencing method. Therefore, the high-throughput sequencing has been applied extensively to genomic analyses, such as screening for mutations, construction of genomic methylation maps, and the study of DNA-protein interactions. Although they have only been available for a short period, high-throughput sequencing techniques are profoundly affecting many of the life sciences, and are opening out new potential avenues of research. With the highly-developed commercial high-throughput sequencing platforms, each laboratory has the opportunity to explore this research field. Therefore, in this paper, we have focused on commercially-popular high-throughput sequencing techniques and the ways in which they have been applied over the past three years. 相似文献
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Christopher P. Middleton Nils Stein Beat Keller Benjamin Kilian Thomas Wicker 《The Plant journal : for cell and molecular biology》2013,73(2):347-356
A 454 sequencing snapshot was utilised to investigate the genome composition and nucleotide diversity of transposable elements (TEs) for several Triticeae taxa, including Triticum aestivum, Hordeum vulgare, Hordeum spontaneum and Secale cereale together with relatives of the A, B and D genome donors of wheat, Triticum urartu (A), Aegilops speltoides (S) and Aegilops tauschii (D). Additional taxa containing the A genome, Triticum monococcum and its wild relative Triticum boeoticum, were also included. The main focus of the analysis was on the genomic composition of TEs as these make up at least 80% of the overall genome content. Although more than 200 TE families were identified in each species, approximately 50% of the overall genome comprised 12–15 TE families. The BARE1 element was the largest contributor to all genomes, contributing more than 10% to the overall genome. We also found that several TE families differ strongly in their abundance between species, indicating that TE families can thrive extremely successfully in one species while going virtually extinct in another. Additionally, the nucleotide diversity of BARE1 populations within individual genomes was measured. Interestingly, the nucleotide diversity in the domesticated barley H. vulgare cv. Barke was found to be twice as high as in its wild progenitor H. spontaneum, suggesting that the domesticated barley gained nucleotide diversity from the addition of different genotypes during the domestication and breeding process. In the rye/wheat lineage, sequence diversity of BARE1 elements was generally higher, suggesting that factors such as geographical distribution and mating systems might play a role in intragenomic TE diversity. 相似文献
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454测序法在环境微生物生态研究中的应用 总被引：3，自引：0，他引：3
传统的Sanger测序技术虽已成熟,但其速度和成本的限制满足不了大规模测序的要求。第二代高通量测序技术结合了乳胶微粒和皮升级反应的454焦磷酸测序法,作为一种高通量测序技术,具有分析结果准确、高速、高灵敏度和高自动化的特点。对454测序法的技术原理和操作步骤进行了介绍,对近年来运用该方法在环境微生物生态研究领域的进展进行了综述。 相似文献
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Raoul Jean Pierre Bonnal Marco Severgnini Alessandra Castaldi Roberta Bordoni Amelia Trimarco Giulio Piluso Gianluigi Condorelli Gianluca De Bellis Vincenzo Nigro 《Analytical biochemistry》2010,406(2):176-184
The X-linked dystrophin gene is well known for its involvement in Duchenne/Becker muscular dystrophies and for its exceptional megabase size. This locus at Xp21 is prone to frequent random molecular changes, including large deletions and duplications, but also smaller variations. To cope with such huge sequence analysis requirements in forthcoming diagnostic applications, we employed the power of the parallel 454 GS-FLX pyrosequencer to the dystrophin locus. We enriched the genomic region of interest by the robust amplification of 62 fragments under universal conditions by the long-PCR protocol yielding 244,707 bp of sequence. Pooled PCR products were fragmented and used for library preparation and DNA sequencing. To evaluate the entire procedure we analyzed four male DNA samples for sequence coverage and accuracy in DNA sequence variation and for any potential bias. We identified 562 known variations and 55 additional variants not yet reported, among which we detected a causative Arg1844Stop mutation in one sample. Sanger sequencing confirmed all changes. Unexpectedly, only 3× coverage was sufficient for 99.9993% accuracy. Our results show that long PCR combined to massive pyrosequencing is very reliable for the analysis of the biggest gene of the human genome and open the doors to other demanding applications in molecular diagnostics. 相似文献
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This study assessed the major histocompatibility complex (MHC) and neutral genetic variation and structure in two percid species, perch Perca fluviatilis and zander Sander lucioperca, in a unique brackish ecosystem, the Baltic Sea. In addition, to assess the importance of MHC diversity to disease susceptibility in these populations, comparisons were made to an introduced, disease susceptible, P. fluviatilis population in Australia. Eighty‐three MHC class II B exon 2 variants were amplified: 71 variants from 92 P. fluviatilis samples, and 12 variants from 82 S. lucioperca samples. Microsatellite and MHC data revealed strong spatial genetic structure in S. lucioperca, but not P. fluviatilis, across the Baltic Sea. Both microsatellite and MHC data showed higher levels of genetic diversity in P. fluviatilis from the Baltic Sea compared to Australia, which may have facilitated the spread of an endemic virus, EHNV in the Australian population. The relatively high levels of genetic variation in the Baltic Sea populations, together with spatial genetic structure, however, suggest that there currently seems to be little risk of disease epidemics in this system. To ensure this remains the case in the face of ongoing environmental changes, fisheries and habitat disturbance, the conservation of local‐scale genetic variation is recommended. 相似文献
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The hyper-variable V4 and V9 regions of the small subunit (SSU) rDNA have been targeted for assessing environmental diversity of microbial eukaryotes using next generation sequencing technologies. Here, we explore how the genetic distances among these short fragments compare with the distances obtained from near full-length SSU-rDNA sequences by comparing all pairwise estimates, as well as within and among species of ciliates. Results show that pairwise distances from V4 more closely match the near full-length SSU-rDNA and are more comparable with previous studies based on much longer SSU-rDNA fragments, then pairwise distances from V9. Thus, studies that use the V4 will estimate similar values of phylotype richness and community structure as would have been estimated using the full-length SSU-rDNA. 相似文献
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李芝李云峰杨之斌唐蜀昆 《现代生物医学进展》2012,12(9):1782-1784
由于传统研究方法成本和速度的限制,远远满足不了对微生物群落大规模的研究,以454测序为代表的新一代高通量测序技术凭借低成本、高通量、流动自动化的优势为研究微生物的多样性和组成提供了新的技术平台。本文就近年来454测序技术在研究人体肠道微生物与疾病关系的应用进行了综述。 相似文献
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Genotyping of multilocus gene families, such as the major histocompatibility complex (MHC), may be challenging because of problems with assigning alleles to loci and copy number variation among individuals. Simultaneous amplification and genotyping of multiple loci may be necessary, and in such cases, next-generation deep amplicon sequencing offers a great promise as a genotyping method of choice. Here, we describe jMHC, a computer program developed for analysing and assisting in the visualization of deep amplicon sequencing data. Software operates on FASTA files; therefore, output from any sequencing technology may be used. jMHC was designed specifically for MHC studies but it may be useful for analysing amplicons derived from other multigene families or for genotyping other polymorphic systems. The program is written in Java with user-friendly graphical interface (GUI) and can be run on Microsoft Windows, Linux OS and Mac OS. 相似文献
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? Premise of the study: Microsatellite markers were developed for the common wetland plant Juncus effusus to assess levels of within- and among-population genetic diversity. ? Methods and Results: Using a next-generation sequencing approach we identified new microsatellite loci, from which 23 were further characterized in a large population of J. effusus. Expected heterozygosity ranged from 0 to 0.64 with a mean of 0.407. Inbreeding coefficients (mean = 0.984) were very high, confirming earlier suggestions of an inbred mating system. Cross-amplification was tested in J. conglomeratus, J. inflexus, J. drummondii, and J. filiformis, with best results in the former. ? Conclusions: The described microsatellite markers will be helpful in assisting studies on, e.g., reproduction, taxonomy, and phylogeography in J. effusus and related species. 相似文献
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Madjid Delghandi Hasifa Afzal Manal Saif Nasser Al Hinai Rafaida Dhuhai Gharib Al-Breiki Dean R. Jerry Hoc Tan Dao 《Animal biotechnology》2016,27(4):310-314
Polymorphic microsatellite loci were isolated for Panulirus ornatus using 454 GS-FLX Titanium pyrosequencing. Fifteen markers containing perfect di-, tri-, tetra-, and penta-nucleotide motifs were consistently co-amplified in five multiplexes in a panel of 91 randomly selected samples. Observed number of alleles varied from 2 to 14 per locus. Observed and expected heterozygosity ranged from 0.090 to 0.79 and 0.08 to 0.87, respectively. Ten loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between 11 loci. The microsatellite markers were also amplified successfully in related Panulirus homarus species with adequate level of polymorphism. The successful cross-species primer amplification of the 15 microsatellites indicates the potential of the developed markers to be transferred to other Panulirus species. The 15 novel microsatellite markers reported in this work add to the previously characterized markers by our group, exhibit adequate levels of polymorphism for wide range of future studies investigating population structure, genetic diversity, and evolutionary relationships among Panulirus species. 相似文献
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? Premise of the study: Microsatellite markers were developed for the invasive plant Microstegium vimineum (Poaceae) to assess its population structure and to facilitate tracking of invasion expansion. ? Methods and Results: Using 454 sequencing, 11 polymorphic and six monomorphic microsatellite primer sets were developed for M. vimineum. The primer sets were tested on individuals sampled from six populations in the United States and China. The polymorphic primers amplified di-, tri-, and tetranucleotide repeats with three to 10 alleles per locus. ? Conclusions: These markers will be useful for a variety of applications including tracking of invasion dynamics and population genetics studies. 相似文献
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The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world. 相似文献
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Jeukens J Boyle B Kukavica-Ibrulj I St-Cyr J Lévesque RC Bernatchez L 《Molecular ecology resources》2011,11(3):541-549
Genomic DNA sequences and other genomic resources are essential towards the elucidation of the genomic bases of adaptive divergence and reproductive isolation. Here, we describe the construction, characterization and screening of a nonarrayed BAC library for lake whitefish (Coregonus clupeaformis). We then show how the combined use of BAC library screening and next-generation sequencing can lead to efficient full-length assembly of candidate genes. The lake whitefish BAC library consists of 181,050 clones derived from a single heterozygous fish. The mean insert size is 92 Kb, representing 5.2 haploid genome equivalents. Ten BAC clones were isolated following a quantitative real-time PCR screening approach that targeted five previously identified candidate genes. Sequencing of these clones on a 454 GS FLX system yielded 178,000 reads with a mean length of 358 bp, for a total of 63.8 Mb. De novo assembly and annotation then allowed retrieval of contigs corresponding to each candidate gene, which also contained up- and/or downstream noncoding sequences. These results suggest that the lake whitefish BAC library combined with next-generation sequencing technologies will be key resources to achieve a better understanding of both adaptive divergence and reproductive isolation in lake whitefish species pairs as well as salmonid evolution in general. 相似文献
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基于限制位点相关的DNA（restriction site associated DNA, RAD）标记的测序方法是一种新型的测序技术.其优点是不仅节省传统测序的试验成本,而且能快速准确的定位出数以千计的基因标记,从而更加适合分子辅助育种的应用.该方法可应用于寻找DNA多态性,鉴别SNP,构建未知基因组序列生物的遗传图谱,定位目的性状基因等.本文主要综述了RAD标记和RAD测序的研究进展及其在分子育种中的应用. 相似文献
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J. Carlsson D. T. Gauthier J. E. L. Carlsson J. P. Coughlan E. Dillane R. D. Fitzgerald U. Keating P. McGinnity L. Mirimin T. F. Cross 《Journal of fish biology》2013,82(3):944-958
By combining next‐generation sequencing technology (454) and reduced representation library (RRL) construction, the rapid and economical isolation of over 25 000 potential single‐nucleotide polymorphisms (SNP) and >6000 putative microsatellite loci from c. 2% of the genome of the non‐model teleost, Atlantic cod Gadus morhua from the Celtic Sea, south of Ireland, was demonstrated. A small‐scale validation of markers indicated that 80% (11 of 14) of SNP loci and 40% (6 of 15) of the microsatellite loci could be amplified and showed variability. The results clearly show that small‐scale next‐generation sequencing of RRL genomes is an economical and rapid approach for simultaneous SNP and microsatellite discovery that is applicable to any species. The low cost and relatively small investment in time allows for positive exploitation of ascertainment bias to design markers applicable to specific populations and study questions. 相似文献
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Chang-Kug Kim Young-Joo Seol Dong-Jun Lee In-Seon Jeong Ung-Han Yoon Gang-Seob Lee Jang-Ho Hahn Dong-Suk Park 《Bioinformation》2013,9(17):887-888
In 2013, National Agricultural Biotechnology Information Center (NABIC) reconstructs a molecular marker
database for useful genetic resources. The web-based marker database consists of three major functional categories:
map viewer, RSN marker and gene annotation. It provides 7250 marker locations, 3301 RSN marker property, 3280
molecular marker annotation information in agricultural plants. The individual molecular marker provides
information such as marker name, expressed sequence tag number, gene definition and general marker information.
This updated marker-based database provides useful information through a user-friendly web interface that assisted
in tracing any new structures of the chromosomes and gene positional functions using specific molecular markers.