共查询到19条相似文献,搜索用时 78 毫秒
1.
目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法. 相似文献
2.
3.
4.
新一代测序技术的研究进展 总被引:3,自引:0,他引:3
大规模DNA测序技术是揭秘人类和其它生物遗传密码的重要技术,在分子生物学和基础医学领域有广泛应用。第二代测序技术的出现使DNA测序的通量大幅提高,测序的成本大幅下降,原来只有在大型测序中心才能完成的测序任务现在已经可以在更多的实验室展开。但是,早期的第二代测序技术仍然存在诸如文库构建过程复杂、测序成本依然较高等缺点。为了克服上述缺点,近三年发展了几种新的第二代和第三代测序技术,这些技术不仅继承了早期第二代测序技术通量高的优点,而且在文库构建等方面取得了重要突破,进一步简化了测序操作,降低了测序成本,缩短了测序时间。本文就几种最新的大规模测序技术的原理、特点与发展趋势进行简要介绍。 相似文献
5.
茄子microRNAs与其靶基因的生物信息学预测 总被引:2,自引:0,他引:2
microRNAs(miRNAs)是一类在真核生物中发现的长度为21 nt左右、非编码、内源性的单链小分子RNA,通过与靶基因的互补发挥转录后水平的负调控作用。目前,已在许多物种中报道了miRNAs的存在,然而还未见关于茄子miRNAs的报道。根据miRNAs在植物中的高度保守性及其前体的二级结构特征,文章通过同源预测的方法,将已知植物的miRNAs与茄子EST数据库比对,经过一系列的筛选,最终预测到12个家族的16条茄子miRNAs,其中包括3个miRNA家族的正义/反义miRNAs,而miR390和miR399家族的正义/反义miRNAs属于第一次发现。文章还通过在线软件psRNATarget预测到15条茄子miRNAs的71个靶基因,这些靶基因主要编码与茄子生长发育、新陈代谢以及胁迫响应等过程相关的蛋白。 相似文献
6.
miRNA(microRNA)是一类小的非编码RNA,普遍存在于动物、植物等多个物种中,研究表明在原生生物以及病毒中也有miRNA。miRNA在生物的成长、发育、细胞凋亡、神经紊乱、癌症等多个方面都起到至关重要的作用,miRNA还可作为Biomarker应用于癌症等疾病的诊断和治疗。miRDOA (miRNA Database and Online Analysis)数据库存储了二百多个物种的miRNA相关信息,提供了基本的浏览和搜索功能。用户可以通过物种来浏览miRNA相关数据,也可以通过miRNA、靶基因或者序列进行检索。除此之外,miRDOA还提供了在线分析模块,主要对高通量测序数据进行初步分析,在此基础上,添加了靶基因预测、表达谱数据差异分析,以及在线BLAST功能。miRDOA是一个完全免费的开源的数据库网站,并实时更新。 相似文献
7.
8.
MicroRNAs(miRNAs)是一类长度约为22nt的内源性非编码RNA,通过与靶基因转录本互补结合调控基因的表达。近年来,研究发现miRNA与癌症发生密切相关,miRNA可以直接充当癌基因或者抑癌基因而影响肿瘤的发生和生长。为更进一步揭示癌症相关miRNA的特征及靶基因的功能,文章通过数据库搜索及文献检索,在人类基因组中发现了475个癌症相关miRNA,系统地比较了癌症相关miRNA与非癌症miRNA以及基因内和基因间区癌症相关miRNA在保守性、SNP位点分布、癌谱及转录调控等特性。研究发现,癌症相关miRNA比非癌症miRNA保守性要强,发生SNP概率比较低,同时发现miRNA所涉及癌症数目与保守性成正相关。基因组定位分析发现,癌症相关miRNA比非癌症miRNA更倾向于成簇存在。进一步对宿主基因、癌症相关miRNA及作用的靶基因与癌症发生进行关联分析,发现一些非癌症miRNA的宿主基因倾向于被癌症miRNA作用。本研究结果为深入理解miRNA与癌症之间的关系,以及进一步为miRNA作为癌症诊断指示物提供理论依据。 相似文献
9.
10.
11.
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
12.
超过50%的耳聋由遗传基因缺陷所致,伴随着基因组学技术的发展,耳聋分子遗传学研究逐渐成为耳科学研究的前沿领域。新一代高通量测序技术的出现,提供了以数据为导向的新的遗传性疾病研究模式,革新了人们对遗传性疾病的认识过程,使得对遗传性疾病的研究策略也发生了重大转变。近年来新一代测序技术(Next generation sequencing,NGS)在耳聋研究中的应用,大大加快了耳聋基因发现的速度,并逐渐向临床应用方向转化。文章总结了遗传性耳聋的特点和研究现状,以及新一代测序技术在耳聋研究中的应用和前景,以及基于NGS技术的耳聋基因研究和临床耳聋基因诊断的发展方向。 相似文献
13.
14.
Oskar Rennstam Rubbmark Daniela Sint Sandra Cupic Michael Traugott 《Molecular ecology resources》2019,19(2):388-399
Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample. 相似文献
15.
运用高通量测序技术分析复杂样品中微生物群落组成及变化趋势,已经成为目前微生物研究领域的热点之一。本研究以复杂土壤样品和应用范围较广的瘤胃食糜样品为对象,选取20、25和30三个扩增循环数分别对样品的16S r RNA基因的V3区进行扩增,然后进行文库构建和测序。最后通过数据分析比较不同的扩增循环数对细菌多样性测定结果的影响。结果表明,扩增循环数越多,捕获到的细菌数量和种类越多;但并非循环数越多,群落中的微生物组成比例最优。整体来看,当扩增循环数为25时,样品中物种的数量和组成是最优的。 相似文献
16.
Jicai Jiang Jiying Wang Haifei Wang Yan Zhang Huimin Kang Xiaotian Feng Jiafu Wang Zongjun Yin Wenbin Bao Qin Zhang Jian-Feng Liu 《BMC genomics》2014,15(1)
Background
Copy number variations (CNVs) confer significant effects on genetic innovation and phenotypic variation. Previous CNV studies in swine seldom focused on in-depth characterization of global CNVs.Results
Using whole-genome assembly comparison (WGAC) and whole-genome shotgun sequence detection (WSSD) approaches by next generation sequencing (NGS), we probed formation signatures of both segmental duplications (SDs) and individualized CNVs in an integrated fashion, building the finest resolution CNV and SD maps of pigs so far. We obtained copy number estimates of all protein-coding genes with copy number variation carried by individuals, and further confirmed two genes with high copy numbers in Meishan pigs through an enlarged population. We determined genome-wide CNV hotspots, which were significantly enriched in SD regions, suggesting evolution of CNV hotspots may be affected by ancestral SDs. Through systematically enrichment analyses based on simulations and bioinformatics analyses, we revealed CNV-related genes undergo a different selective constraint from those CNV-unrelated regions, and CNVs may be associated with or affect pig health and production performance under recent selection.Conclusions
Our studies lay out one way for characterization of CNVs in the pig genome, provide insight into the pig genome variation and prompt CNV mechanisms studies when using pigs as biomedical models for human diseases.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-593) contains supplementary material, which is available to authorized users. 相似文献17.
18.
Lik Wee Lee Shile Zhang Alton Etheridge Li Ma Dan Martin David Galas Kai Wang 《RNA (New York, N.Y.)》2010,16(11):2170-2180
MicroRNAs (miRNAs) have been implicated to play key roles in normal physiological functions, and altered expression of specific miRNAs has been associated with a number of diseases. It is of great interest to understand their roles and a prerequisite for such study is the ability to comprehensively and accurately assess the levels of the entire repertoire of miRNAs in a given sample. It has been shown that some miRNAs frequently have sequence variations termed isomirs. To better understand the extent of miRNA sequence heterogeneity and its potential implications for miRNA function and measurement, we conducted a comprehensive survey of miRNA sequence variations from human and mouse samples using next generation sequencing platforms. Our results suggest that the process of generating this isomir spectrum might not be random and that heterogeneity at the ends of miRNA affects the consistency and accuracy of miRNA level measurement. In addition, we have constructed a database from our sequencing data that catalogs the entire repertoire of miRNA sequences (http://galas.systemsbiology.net/cgi-bin/isomir/find.pl). This enables users to determine the most abundant sequence and the degree of heterogeneity for each individual miRNA species. This information will be useful both to better understand the functions of isomirs and to improve probe or primer design for miRNA detection and measurement. 相似文献
19.
Caroline K. Glidden Anson V. Koehler Ross S. Hall Muhammad A. Saeed Mauricio Coppo Brianna R. Beechler Bryan Charleston Robin B. Gasser Anna E. Jolles Abdul Jabbar 《Ecology and evolution》2020,10(1):70-80
- Increasing access to next‐generation sequencing (NGS) technologies is revolutionizing the life sciences. In disease ecology, NGS‐based methods have the potential to provide higher‐resolution data on communities of parasites found in individual hosts as well as host populations.
- Here, we demonstrate how a novel analytical method, utilizing high‐throughput sequencing of PCR amplicons, can be used to explore variation in blood‐borne parasite (Theileria—Apicomplexa: Piroplasmida) communities of African buffalo at higher resolutions than has been obtained with conventional molecular tools.
- Results reveal temporal patterns of synchronized and opposite fluctuations of prevalence and relative abundance of Theileria spp. within the host population, suggesting heterogeneous transmission across taxa. Furthermore, we show that the community composition of Theileria spp. and their subtypes varies considerably between buffalo, with differences in composition reflected in mean and variance of overall parasitemia, thereby showing potential to elucidate previously unexplained contrasts in infection outcomes for host individuals.
- Importantly, our methods are generalizable as they can be utilized to describe blood‐borne parasite communities in any host species. Furthermore, our methodological framework can be adapted to any parasite system given the appropriate genetic marker.
- The findings of this study demonstrate how a novel NGS‐based analytical approach can provide fine‐scale, quantitative data, unlocking opportunities for discovery in disease ecology.