共查询到20条相似文献，搜索用时 15 毫秒
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Wendy Wang Wei X. Tan Denis Bertrand Amanda H. Q. Ng Esther J. H. Boey Jayce J. Y. Koh Niranjan Nagarajan Rudolf Meier 《Molecular ecology resources》2018,18(5):1035-1049
DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well‐equipped molecular laboratory and is time‐consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION? and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION? reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch‐free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION? runs that represent three different stages of MinION? development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION? barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION? barcodes have an accuracy of 99.3%–100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be 相似文献
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Seitaro Denbo Katsutoshi Aono Takaaki Kai Rei Yagasaki Iñaki Ruiz-Trillo Hiroshi Suga 《Development, growth & differentiation》2019,61(1):34-42
The genome sequences of unicellular holozoans, the closest relatives to animals, are shedding light on the evolution of animal multicellularity, shaping the genetic contents of the putative premetazoans. However, the assembly quality of the genomes remains poor compared to the major model organisms such as human and fly. Improving the assembly is critical for precise comparative genomics studies and further molecular biological studies requiring accurate sequence information such as enhancer analysis and genome editing. In this report, we present a new strategy to improve the assembly by fully exploiting the information of Illumina mate-pair reads. By visualizing the distance and orientation of the mapped read pairs, we could highlight the regions where possible assembly errors exist in the genome sequence of Capsaspora, a lineage of unicellular holozoans. Manual modification of these errors repaired 590 assembly problems in total and reassembled 84 supercontigs into 55. Our telomere prediction analysis using the read pairs containing the pan-eukaryotic telomere-like sequence identified at least 13 chromosomes. The resulting new assembly posed us a re-annotation of 112 genes, including 15 putative receptor protein tyrosine kinases. Our strategy thus provides a useful approach for improving assemblies of draft genomes, and the new Capsaspora genome offers us an opportunity to adjust the view on the genome of the unicellular animal ancestor. 相似文献
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本研究介绍了基因组结构变异检测的生物信息学基本方法和前沿技术。对基于第二代测序技术的四种检测方法(读对方法,读深方法,分裂片段方法和序列拼接方法)的原理和特点进行了详细解读,分析了第二代测序技术应用在检测结构变异上的特点与发展趋势。最后介绍了三代测序、Linked-reads和光学物理图谱等新技术在基因组结构变异检测中的应用,论述了融合新技术的结构变异检测方法的特点与优势。 相似文献
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Although the diploid nature has been observed for over 50 years, phasing the diploid is still a laborious task. The speed and throughput of next generation sequencing have largely increased in the past decades. However, the short read-length remains one of the biggest challenges of haplotype analysis. For instance, reads as short as 150 bp span no more than one variant in most cases. Numerous experimental technologies have been developed to overcome this challenge. Distance, complexity and accuracy of the linkages obtained are the main factors to evaluate the efficiency of whole genome haplotyping methods. Here, we review these experimental technologies, evaluating their efficiency in linkages obtaining and system complexity. The technologies are organized into four categories based on its strategy: (i) chromosomes separation, (ii) dilution pools, (iii) crosslinking and proximity ligation, (ix) long-read technologies. Within each category, several subsections are listed to classify each technology. Innovative experimental strategies are expected to have high-quality performance, low cost and be labor-saving, which will be largely desired in the future. 相似文献
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Xiaoling Yu Wenqian Jiang Yang Shi Hanhui Ye Jun Lin 《Journal of cellular and molecular medicine》2019,23(11):7143-7150
Infectious diseases are a type of disease caused by pathogenic microorganisms. Although the discovery of antibiotics changed the treatment of infectious diseases and reduced the mortality of bacterial infections, resistant bacterial strains have emerged. Anti‐infective therapy based on aetiological evidence is the gold standard for clinical treatment, but the time lag and low positive culture rate of traditional methods of pathogen diagnosis leads to relative difficulty in obtaining the evidence of pathogens. Compared with traditional methods of pathogenic diagnosis, next‐generation and third‐generation sequencing technologies have many advantages in the detection of pathogenic microorganisms. In this review, we mainly introduce recent progress in research on pathogenic diagnostic technology and the applications of sequencing technology in the diagnosis of pathogenic microorganisms. This review provides new insights into the application of sequencing technology in the clinical diagnosis of microorganisms. 相似文献
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F. Nadiya N. Anjali J. Thomas A. Gangaprasad K. K. Sabu 《Plant biology (Stuttgart, Germany)》2019,21(1):3-14
- Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
- In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
- We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
- Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.
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Tom Oosting Elena Hilario Maren Wellenreuther Peter A. Ritchie 《Ecology and evolution》2020,10(16):8643-8651
The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications. 相似文献
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Cédric Mariac Nora Scarcelli Juliette Pouzadou Adeline Barnaud Claire Billot Adama Faye Ayite Kougbeadjo Vincent Maillol Guillaume Martin François Sabot Sylvain Santoni Yves Vigouroux Thomas L. P. Couvreur 《Molecular ecology resources》2014,14(6):1103-1113
Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large data sets thanks to next‐generation sequencing methods. In this study, we develop an easy and cost‐effective protocol for in‐solution enrichment hybridization capture of complete chloroplast genomes applicable at deep‐multiplexed levels. The protocol uses cheap in‐house species‐specific probes developed via long‐range PCR of the entire chloroplast. Barcoded libraries are constructed, and in‐solution enrichment of the chloroplasts is carried out using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes. 相似文献
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高通量测序技术的快速发展对食品微生物发酵过程和机制研究产生了深刻的影响,主要体现在食品微生物生理功能、代谢能力和进化的研究以及食品微生物群落结构、动态变化及其对环境的响应机制等方面。另外,通过对食品微生物基因组和元基因组进行数据分析,也对食品发酵过程优化、微生物功能改造、食源性微生物疾病预防和控制等提供了重要的依据。本文总结了近年来利用高通量测序技术对食品微生物基因组和元基因组进行测序的研究,并探讨了测序技术的发展对食品微生物研究的影响及发展趋势。 相似文献
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Clio Der Sarkissian Morten E. Allentoft María C. ávila-Arcos Ross Barnett Paula F. Campos Enrico Cappellini Luca Ermini Ruth Fernández Rute da Fonseca Aurélien Ginolhac Anders J. Hansen Hákon Jónsson Thorfinn Korneliussen Ashot Margaryan Michael D. Martin J. Víctor Moreno-Mayar Maanasa Raghavan Morten Rasmussen Marcela Sandoval Velasco Hannes Schroeder Mikkel Schubert Andaine Seguin-Orlando Nathan Wales M. Thomas P. Gilbert Eske Willerslev Ludovic Orlando 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2015,370(1660)
The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field''s focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past. 相似文献
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目的 探索江苏常熟地区汉族人群在47个常染色体微单倍型基因座的遗传多态性,评估应用效能及法庭科学参数。方法 采用MHSeqTyper47混合DNA鉴定试剂盒进行基因座复合扩增及文库构建,使用MiSeq FGx测序平台进行测序,测得的数据应用MHTyper数据分析软件进行分析,对获得的样本遗传信息进行评估,结合千人基因组数据（1000 Genomes Project phase 3,1KG）评估群体间遗传分化指数及遗传距离,并计算法庭科学参数。结果 江苏常熟地区汉族人群与1KG中的中国北京群体的遗传分化和遗传距离最小,并得到最接近的有效等位基因数（Ae）,累积随机匹配概率（combined matching probability,CMP）与1KG中东亚参考人群的5个群体均接近,为1.25×10-36,累积非父排除概率达0.999 999 999 964 1。结论 本研究报告了47个微单倍型基因座在江苏常熟地区汉族人群中的等位基因频率及遗传多态性信息,为47个微单倍型在法医学应用中提供了数据基础。另外,比较了1KG参考人群与江苏常熟地区汉族人群的多态性差异,并揭示了47个微单倍型在江苏常熟地区汉族人群中的遗传结构。总的来说,1KG中的东亚人群参考数据更符合江苏常熟地区汉族人群的遗传特征。 相似文献
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Background: The Oxford MinION nanopore sequencer is the recently appealing third-generation genome sequencing device that is portable and no larger than a cellphone. Despite the benefits of MinION to sequence ultra-long reads in real-time, the high error rate of the existing base-calling methods, especially indels (insertions and deletions), prevents its use in a variety of applications. Methods: In this paper, we show that such indel errors are largely due to the segmentation process on the input electrical current signal from MinION. All existing methods conduct segmentation and nucleotide label prediction in a sequential manner, in which the errors accumulated in the first step will irreversibly influence the final base-calling. We further show that the indel issue can be significantly reduced via accurate labeling of nucleotide and move labels directly from the raw signal, which can then be efficiently learned by a bi-directional WaveNet model simultaneously through feature sharing. Our bi-directional WaveNet model with residual blocks and skip connections is able to capture the extremely long dependency in the raw signal. Taking the predicted move as the segmentation guidance, we employ the Viterbi decoding to obtain the final base-calling results from the smoothed nucleotide probability matrix. Results: Our proposed base-caller, WaveNano, achieves good performance on real MinION sequencing data from Lambda phage. Conclusions: The signal-level nanopore base-caller WaveNano can obtain higher base-calling accuracy, and generate fewer insertions/deletions in the base-called sequences. 相似文献
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插入位点分析对于金针菇功能基因组学的研究极为重要,分析方法常用反向PCR、热不对称交错PCR、Tail-PCR、染色体步移等,存在操作复杂、消耗时间长、特异性较差、效率低等缺点。近年来开始应用基因组重测序的方法,对转化子逐一测序与分析,工作量较大、费用较高。本研究应用矩阵设计,把多个转化子的DNA混合构成样品池,重测序后分析插入位点,M个样品池的测序数据可分析M×(M+1)/2个转化子的插入位点。应用矩阵设计构建6个样品池检测21个转化子,获得21个插入位点,表明这种方法可行、适合大样本分析,如突变体库。 相似文献