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A platform for massive parallel sequencing of longPCR amplicons

longPCR 扩增子大规模并行测序平台

基本信息

批准号：
BB/H023534/1
负责人：
David Littlewood
金额：
$ 15.26万
依托单位：
Natural History Museum
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2010
资助国家：
英国
起止时间：
2010 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FH023534%2F1
关键词：
platform massive parallel sequencing longPCR

项目摘要

New generation sequencing techniques offer an unprecedented means of sequencing genes and genomes at a fraction of previous costs and at a phenomenal density of coverage. A variety of platforms offer different techniques. 454 pyrosequencing, also known as massive parallel sequencing, has the advantage of providing relatively long sequence reads (~450 nucleotides) in over 1 million individual reaction chambers on a pico-titre plate; developments are under way to capture even longer reads. When mixing templates from different sources there is a need to link sequences with their source. Two ways are possible and include (i) processing individual samples on single pico-titre plates or individual gasketed sections of a plate (up to 16), or (ii) chemically-tagging templates with unique sample-specific markers. Long lengths of DNA (up to 20,000 nucleotides) are routinely amplified with specialised polymerase chain reactions for a diversity of purposes by a wide variety of users of molecular tools. By sequencing the ends of these long amplicons using traditional methods, and by relying on bioinformatic tools to accurately unscramble the data, we propose a method that allows hundreds of long amplicons to be pooled, fragmented, massively parallel sequenced, accurately reassembled and identified, thus reducing existing costs by orders of magnitude. The technique will allow routine multiplex sequencing of longPCRs where only short fragments could be sequenced previously, or where expensive sample-specific tagging and/or cloning was required. We will test the methodology by generating longPCR amplicons from parasitic helminths, for which: (i) we have a wide diversity of samples available and considerable experience of handling, (ii) there is wide interest and need, including diagnostics, biodiversity studies and evolutionary parasitology. Simulation studies will be used in conjunction with real data to develop, refine and test the bioinformatics pipeline for wider application. The methodology and associated open access computer applications will be transferable to any biological system where diverse longPCR fragments are sequenced regardless of the origin of the DNA.

新一代测序技术提供了一种前所未有的基因和基因组测序手段，其成本仅为以前的一小部分，覆盖范围也达到了惊人的密度。各种平台提供不同的技术。454焦磷酸测序，也被称为大规模平行测序，具有在微滴板上超过100万个单独的反应室中提供相对较长的序列读取（约450个核苷酸）的优势；人们正在开发更长的阅读方式。当混合来自不同源的模板时，需要将序列与其源链接起来。有两种可能的方法，包括(i)在单个微滴度板或板的单个垫圈部分（最多16个）上处理单个样品，或（ii）用独特的样品特异性标记物对模板进行化学标记。长长度的DNA（多达20,000个核苷酸）通常通过专门的聚合酶链反应进行扩增，用于各种各样的分子工具用户。通过使用传统方法对这些长扩增子的末端进行测序，并依靠生物信息学工具准确地解读数据，我们提出了一种方法，该方法允许数百个长扩增子汇集，碎片化，大规模并行测序，准确地重新组装和识别，从而降低了现有成本的数量级。该技术将允许对以前只能测序短片段的长pcr进行常规多重测序，或者需要昂贵的样品特异性标记和/或克隆。我们将通过从寄生蠕虫中产生长pcr扩增子来测试该方法，为此：(i)我们有广泛的可用样品和丰富的处理经验，（ii）有广泛的兴趣和需求，包括诊断，生物多样性研究和进化寄生虫学。模拟研究将与实际数据结合使用，以开发、改进和测试生物信息学管道，以便更广泛地应用。该方法和相关的开放获取计算机应用程序将可转移到任何生物系统中，无论DNA的起源如何，都可以对各种长pcr片段进行测序。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The mitochondrial genome of Parascaris univalens--implications for a "forgotten" parasite.

帕斯卡里斯单相的线粒体基因组 - 寄生虫“被遗忘”的象征。

DOI：
10.1186/1756-3305-7-428
发表时间：
2014-09-04
期刊：
Parasites & vectors
影响因子：
3.2
作者：
Jabbar A;Littlewood DT;Mohandas N;Briscoe AG;Foster PG;Müller F;von Samson-Himmelstjerna G;Jex AR;Gasser RB
通讯作者：
Gasser RB

Assessment of the genetic relationship between Dictyocaulus species from Bos taurus and Cervus elaphus using complete mitochondrial genomic datasets.

DOI：
10.1186/1756-3305-5-241
发表时间：
2012-10-30
期刊：
Parasites & vectors
影响因子：
3.2
作者：
Gasser RB;Jabbar A;Mohandas N;Höglund J;Hall RS;Littlewood DT;Jex AR
通讯作者：
Jex AR

Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal health significance.

DOI：
10.1186/1471-2164-11-447
发表时间：
2010-07-22
期刊：
BMC genomics
影响因子：
4.4
作者：
Jia WZ;Yan HB;Guo AJ;Zhu XQ;Wang YC;Shi WG;Chen HT;Zhan F;Zhang SH;Fu BQ;Littlewood DT;Cai XP
通讯作者：
Cai XP

Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species.