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在过去的30年中,作为最重要的生物医学研究手段之一,DNA测序技术的数据产出能力呈指数增长,而且这一技术本身也演变成为一个面向工程学和物理学的新技术领域.本文分析了下一代测序仪的技术特点,并对其未来的发展以及应用进行了前瞻性的展望.预期在刚刚出现的技术中,有些技术假以时日能够发展成熟,实现1000美元基因组和100美元基因组的目标.同时建议中国科学家在这场对科学研究以及社会医疗保健体系都将产生深远影响的运动中发挥积极的作用. 相似文献
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遗传性心肌病(Inherited cardiomyopathy, ICM)是一种常见的遗传性心脏疾病,主要由基因突变所致,是青少年和年轻运动员猝死的最主要原因之一。到目前为止,已经发现约100个基因和其致病有关,这些基因相关的变异位点具有不同的致病机制。随着临床遗传检测在遗传疾病诊断中的应用,对遗传性心肌病的分子遗传学特性及其致病机制进行深入了解,是对该病遗传诊断的关键。下一代半导体测序仪在2010年底由美国Life Technologies公司发布,其以布满微孔的高密度半导体芯片为测序基础,具有快速、经济、灵敏性好、准确率高等特点,已经应用于遗传疾病的突变筛查。文章主要对遗传性心肌病的分子遗传学特性和下一代半导体测序技术在遗传性心肌病遗传检测中的应用以及面临的挑战进行了概括总结,有助于遗传性心肌病的诊断、预防和治疗。 相似文献
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高通量测序技术及其应用 总被引:14,自引:0,他引:14
高通量测序技术是DNA测序发展历程的一个里程碑,它为现代生命科学研究提供了前所未有的机遇。详细介绍了以454、Solexa和SOLiD为代表的第二代高通量测序技术,以HeliScope TIRM和Pacific Biosciences SMRT为代表的单分子测序技术,以及最近Life Science公司推出的Ion Personal Genome Machine (PGM)测序技术等高通量测序技术的最新进展。在此基础上,阐述了高通量测序技术在基因组测序、转录组测序、基因表达调控、转录因子结合位点的检测以及甲基化等研究领域的应用。最后,讨论了高通量测序技术在成本和后续数据分析等方面存在的问题及其未来的发展前景。 相似文献
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下一代测序技术在表观遗传学研究中的重要应用及进展 总被引:1,自引:0,他引:1
下一代测序技术(Next generation sequencing,NGS)的出现,极大地促进了表观遗传学的研究。将NGS技术引入表观遗传学,便形成了以NGS为基础的各种表观遗传学测序及研究方法,如:全基因组亚硫酸氢盐测序法(Whole genome bisulfite sequencing,WGBS)、简化代表性亚硫酸氢盐测序法(Reduced representation bisulfite sequencing,RRBS)、甲基化DNA免疫共沉淀测序(Methylated DNA immunoprecipitationsequencing,MeDIP-seq)、染色质免疫共沉淀测序(Chromatin immunoprecipitation-sequencing,ChIP-seq)、Tet辅助重亚硫酸盐测序法(Tet-assisted bisulfite sequencing,TAB-seq)、各种染色体构象捕获测序(Chromosome conformation capture sequencing,3C-seq)技术、DnaseⅠ-seq/MNase-seq/FAIRE-seq以及RNA测序(RNA sequencing,RNA-seq)。这些方法的应用和普及改变了人们对多种表观遗传现象的传统认识,使研究人员能够更加全面地深入了解各种表观遗传标志在机体内的广泛分布,以及如何在外界因素的影响下发生相应的动态变化。文章概述了当今主要商业NGS平台的原理和特点,系统介绍了以NGS方法为基础衍生出来的各种表观遗传学测序及研究方法,并在此基础上对近年来应用NGS技术在表观遗传学研究领域中取得的最新研究成果进行了综述。 相似文献
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下一代测序中ChIP-seq数据的处理与分析 总被引:1,自引:0,他引:1
将染色质免疫共沉淀技术(ChIP)与下一代高通量测序技术相结合的染色质免疫共沉淀测序(ChIP-seq),已成为功能基因组学、特别是基因表达调控领域研究的关键技术。ChIP-seq实验带来的海量数据向生物信息学研究人员提出了新的挑战。由于此领域数据处理技术的发展大大滞后于实验技术进步,有必要系统地介绍和回顾ChIP-seq数据处理的各个方面,以便更多研究人员进入此领域设计或改进相应的算法。文章结合实例详细介绍了ChIP-seq数据整个流程,并重点讨论了其中的主要问题和关键环节,为这一研究领域的科研人员提供一个快速而深入的认识。 相似文献
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目的:针对下一代测序数据,尤其是单端测序数据,研究快速、准确查找Indel的方法。方法:先与全基因组参考序列进行快速比对,筛选出包含Indel的序列;再对这些序列进行双向的二次比对,确定Indel长度;最后借助长度信息在锁定范围内查找Indel的确切位置和相关信息。结果:本文成功构建FIND(Fast INDel detection system)系统,用于从单端测序数据中查找Indel信息。以模拟测序数据作为测试数据,在12X测试数据情况下,FIND的灵敏度和特异性分别为87.71%和99.66%,而且该性能还随着测序倍数的增加而提升。结论:充分利用比对过程获取的信息,在确定Indle长度的同时也确定出其大致位置,最终在局部范围内实现对单端测序数据中Indle的快速而准确的查找。 相似文献
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Olivier Harismendy Pauline C Ng Robert L Strausberg Xiaoyun Wang Timothy B Stockwell Karen Y Beeson Nicholas J Schork Sarah S Murray Eric J Topol Samuel Levy Kelly A Frazer 《Genome biology》2009,10(3):R32-13
Background
Next generation sequencing (NGS) platforms are currently being utilized for targeted sequencing of candidate genes or genomic intervals to perform sequence-based association studies. To evaluate these platforms for this application, we analyzed human sequence generated by the Roche 454, Illumina GA, and the ABI SOLiD technologies for the same 260 kb in four individuals.Results
Local sequence characteristics contribute to systematic variability in sequence coverage (>100-fold difference in per-base coverage), resulting in patterns for each NGS technology that are highly correlated between samples. A comparison of the base calls to 88 kb of overlapping ABI 3730xL Sanger sequence generated for the same samples showed that the NGS platforms all have high sensitivity, identifying >95% of variant sites. At high coverage, depth base calling errors are systematic, resulting from local sequence contexts; as the coverage is lowered additional 'random sampling' errors in base calling occur.Conclusions
Our study provides important insights into systematic biases and data variability that need to be considered when utilizing NGS platforms for population targeted sequencing studies. 相似文献13.
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P.?A.?Zhulidov E.?A.?Bogdanova A.?S.?Shcheglov I.?A.?Shagina L.?L.?Wagner G.?L.?Khazpekov V.?V.?Kozhemyako S.?A.?Lukyanov
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, effectively cleaves double-stranded DNA, and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935–1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 186–194.Original Russian Text Copyright © 2005 by Zhulidov, Bogdanova, Shcheglov, Shagina, Wagner, Khazpekov, Kozhemyako, Lukyanov, Shagin. 相似文献
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Sulonen AM Ellonen P Almusa H Lepistö M Eldfors S Hannula S Miettinen T Tyynismaa H Salo P Heckman C Joensuu H Raivio T Suomalainen A Saarela J 《Genome biology》2011,12(9):R94-18
Background
Techniques enabling targeted re-sequencing of the protein coding sequences of the human genome on next generation sequencing instruments are of great interest. We conducted a systematic comparison of the solution-based exome capture kits provided by Agilent and Roche NimbleGen. A control DNA sample was captured with all four capture methods and prepared for Illumina GAII sequencing. Sequence data from additional samples prepared with the same protocols were also used in the comparison.Results
We developed a bioinformatics pipeline for quality control, short read alignment, variant identification and annotation of the sequence data. In our analysis, a larger percentage of the high quality reads from the NimbleGen captures than from the Agilent captures aligned to the capture target regions. High GC content of the target sequence was associated with poor capture success in all exome enrichment methods. Comparison of mean allele balances for heterozygous variants indicated a tendency to have more reference bases than variant bases in the heterozygous variant positions within the target regions in all methods. There was virtually no difference in the genotype concordance compared to genotypes derived from SNP arrays. A minimum of 11× coverage was required to make a heterozygote genotype call with 99% accuracy when compared to common SNPs on genome-wide association arrays.Conclusions
Libraries captured with NimbleGen kits aligned more accurately to the target regions. The updated NimbleGen kit most efficiently covered the exome with a minimum coverage of 20×, yet none of the kits captured all the Consensus Coding Sequence annotated exons. 相似文献18.
MicroRNAs(miRNAs)是一类在进化上高度保守的非编码小分子单链RNA(~22nt),在基因转录后调控中发挥至关重要的作用。越来越多的证据表明,miRNAs参与很多重要的生理和病理过程,例如发育、器官形成、调亡、细胞增殖、肿瘤发生等。近年来飞速发展的新一代测序技术在miRNA检测方面具有重要的应用。文章简要介绍了新一代测序技术3大平台的基本步骤和原理,测序数据的生物信息学分析方法以及新一代测序技术在miRNA方向的主要应用。相比于传统的miRNA检测方法,新一代测序技术具有通量高、对遗传物质检测完全且准确度高,可重复性好等优点,在探索新miRNA、miRNA互补链、miRNA编辑、miRNA异构体检测以及miRNA靶基因检测等方面具有巨大优势。随着新一代测序技术的不断发展,测序成本不断降低,在未来几年,新一代测序技术的使用率或将大大增加。新一代测序技术的不断应用将进一步促进人类对于miRNA在各种生理病理过程中的功能和调控的认识。 相似文献
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文章旨在建立一种基因组目标靶序列捕捉文库的方法,并结合第二代测序技术,以实现候选基因区段的深度测序。利用Agilent公司的eArray在线平台,对1250个基因的11824个外显子共2414977bp的基因组序列进行120个碱基长度的捕捉探针(钓饵)设计,并制备成SureSelect液相靶序列捕获试剂。选用2例人基因组DNA,超声打断后末端补平并磷酸化,连接SOLiD接头,回收150bp~200bp的DNA片段,与靶序列探针杂交捕获目标序列,油包水微乳滴PCR扩增后,磁珠分离富集,上SOLiD测序系统通过工作流程分析(WFA)进行文库质量的评价,或正式测序反应。结果显示对所包含的11147个基因外显子片段设计出并合成了46509个捕捉探针,制备成SureSelect试剂盒。探针可有效地捕捉并富集基因组DNA的目标靶片段,定量PCR显示富集效率可达29倍。WFA分析表明文库可以在SOLiD仪器进行正式测序。测序结果显示靶序列区域的测序数占有效总测序数的比例达到70%,覆盖率均在200×以上。结果表明本研究所建立的SureSelect基因组靶序列捕捉、富集建立测序文库的技术路线可行,可直接用于SOLiD测序仪的测序。 相似文献
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Summary Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized. 相似文献